Antibodies reactive with an epitope located in the n-terminal region of muc5ac comprising cysteine-rich subdomain 2 (cys2)

ABSTRACT

The present invention concerns compositions and methods of use of antibodies or antibody fragments that bind to an epitope located within the second cysteine-rich domain (Cys2, amino acid residues 1575-1725) of MUC5AC. The antibodies bind with high specificity and selectivity to pancreatic cancer and are of use for therapy, detection and/or diagnosis of pancreatic cancer. In preferred embodiments, therapeutic antibody may be conjugated to at least one therapeutic agent, such as  90 Y. Both in vivo and in vitro detection of pancreatic cancer may be performed with the subject methods and compositions. Specific dosages of radiolabeled antibody and/or gemcitabine, of use in human pancreatic cancer patients, are disclosed herein.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/753,249, filed Jun. 29, 2015, which was a continuation-in-part ofU.S. patent application Ser. No. 14/632,480 (now U.S. Pat. No.9,238,084) filed Feb. 26, 2015, which was a divisional of U.S. patentapplication Ser. No. 14/242,138 (now U.S. Pat. No. 9,005,613), filedApr. 1, 2014, which claimed the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Application Ser. No. 61/807,176, filed Apr. 1,2013, 61/818,708, filed May 2, 2013, and 61/896,909, filed Oct. 29,2013. This application claims the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Application Ser. No. 62/018,989, filed Jun. 30,2014, 62/091,932, filed Dec. 15, 2014, and 62/148,428, filed Apr. 16,2015, the entire text of each priority application incorporated hereinby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 16, 2015, isnamed IMM352US1_SL.txt and is 56,774 bytes in size.

FIELD OF THE INVENTION

This invention relates to anti-pancreatic cancer antibodies andantigen-binding fragments thereof that bind to MUC5AC mucin inpancreatic cancer. Preferably, the antibodies or fragments thereof bindto an epitope located within the second to fourth cysteine-richsubdomains of MUC5AC (amino acid residues 1575-2052, Cys2-Cys4). Morepreferably, the antibodies bind to an epitope located in amino acidresidues 1575-1725 and 1903-2052 (Cys2 and Cys4). Even more preferably,the antibodies bind to an epitope located in amino acid residues1575-1725 (Cys2+). Most preferably, the antibodies bind to an epitopelocated in the Cys2 subdomain of MUC5AC. In preferred embodiments, theanti-pancreatic cancer antibody binds to the same epitope as, orcompetes for binding to MUC5AC with a PAM4 antibody comprising the lightchain variable region complementarity-determining region (CDR) sequencesCDR1 (SASSSVSSSYLY, SEQ ID NO: 1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3(HQWNRYPYT, SEQ ID NO:3); and the heavy chain CDR sequences CDR1 (SYVLH,SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3(GFGGSYGFAY, SEQ ID NO:6). The subject antibodies or antibody fragmentsbind with high selectivity to pancreatic cancer cells to allow detectionand/or diagnosis of pancreatic adenocarcinoma at the earliest stages ofthe disease. Most preferably, antibody-based assays are capable ofdetecting about 85% or more of pancreatic adenocarcinomas, with a falsepositive rate of about 5% or less for healthy controls. In particularembodiments, the methods and compositions can be used to detect and/ordiagnose pancreatic adenocarcinoma by screening serum samples fromsubjects and preferably can detect 60% or more of stage I pancreaticcancers and 80% or more of stage II cancers by serum sample analysis. Instill other embodiments, reactivity with the anti-pancreatic cancerantibody can be used to detect occult pancreatic cancer or neoplasticprecursor lesions against a background of pancreatitis or benignpancreatic hyperplasia.

The anti-pancreatic cancer antibody is of use for diagnosis, detectionand/or treatment of pancreatic cancer. For therapy, the antibody orfragment thereof may be administered as a naked antibody, as an antibodycomplex, as an antibody fusion protein, or conjugated to at least onetherapeutic agent, such as a radionuclide, an immunomodulator, ahormone, a hormone antagonist, an enzyme, an oligonucleotide such as ananti-sense oligonucleotide or a siRNA, an enzyme inhibitor, aphotoactive therapeutic agent, a cytotoxic agent such as a drug ortoxin, an angiogenesis inhibitor and a pro-apoptotic agent. Mostpreferably, the antibody is a ⁹⁰Y-hPAM4 antibody and the radiolabeledantibody may be administered in fractionated dosages for treatingpancreatic cancer.

BACKGROUND

The number of patients who succumb to pancreatic ductal adenocarcinoma(PDAC) each year continues to rise, unlike other leading cancers wheresurveillance and/or screening technologies have led to a decrease incancer-related mortality rates (Cardin & Berlin, 2013, J Natl CancerInst 105:1675-6; Ma et al., 2013, J Natl Cancer Inst 105:1694-1700;Siegel et al. 2012, Cancer statistics, 62:10-29). Unfortunately, themortality rate for PDAC is nearly equal to the incidence. The overallsurvival rate for all stages of pancreatic cancer diagnosed between 2001and 2007 is only 20% after one year, and about 6% after 5 years (Siegelet al. 2012, Cancer statistics, 62:10-29). With the alarming increase inPDAC incidence, it is projected that by the year 2030, pancreatic cancerwill become the second leading cause of cancer deaths in the UnitedStates (Rahib et al., 2014, Cancer Res 74:2913-21). The major reason forthis poor prognosis is the inability to detect the disease at an earlystage, when curative measures may have a greater opportunity to providesuccessful outcomes.

Biomarkers, whether they are biological, chemical, or physical innature, have proven of significant value in providing informationleading to the earlier detection and diagnosis of cancer, such as breast(Goldhirsch et al., 2003, Ann Oncol 14:1212-4), colon (Mandel et al.,1993, N Engl J Med 328:1365-71), and prostate (Jacobsen et al., 1995,JAMA 274:1445-9), resulting in improved patient outcomes. Unfortunately,this has not been the case for PDAC. Despite considerable attentiondirected towards discovery of biomarkers for PDAC (Lennon et al., 2014,Cancer Research 74:1-9), to date no FDA-approved means for earlydetection/diagnosis exists. A need exists for more effectivecompositions and methods for early detection and/or diagnosis ofprostate cancer, preferably at the earliest stages of the disease.

In addition to more effective and earlier means of detection, a needalso exists for better therapeutic treatments for pancreatic cancer. Theoutlook for patients with advanced pancreatic adenocarcinoma remainspoor (Hidalgo, 2010, N Engl J Med 362:1605-17). In the frontline, mediansurvival was 6.2-6.7 months with gemcitabine alone (Burris et al., 1997,J Clin Oncol 15:2403-13) or with erlotinib (Moore et al., 2007, J ClinOncol 25:1960-6), 8.5 months combined with albumin-bound paclitaxel (VonHoff et al., 2013, N Engl J Med 369:1691-1703), and 11.1 months forthose able to tolerate combination chemotherapy (FOLFIRINOX) (Conroy etal., 2011, N Engl J Med 364:1817-25). Beyond 1st line, the survivaladvantage with chemotherapy remains limited (Rahma et al., 2013, AnnOncol 24:1972-9; Oettle et al., 2014, J Clin Oncol 32:2423-9) and aftertwo prior treatments (one usually gemcitabine-based, the otherfluoropyrimidine-based), there are no accepted treatments (Seufferleinet al., 2012, Ann Oncol 23(suppl 7):vii33-40; Almhanna & Kim, 2008,Oncology (Williston Park) 22:1176-83).

There is an unmet need for more effective therapies in pancreatic cancerpatients who have received and shown resistance to or relapsed from twoor more prior therapies.

SUMMARY

In various embodiments, the present invention concerns antibodies,antigen-binding antibody fragments and fusion proteins that bind to theMUC5AC pancreatic cancer mucin. Preferably, the antibodies or fragmentsthereof bind to an epitope located within the second to fourthcysteine-rich subdomains of MUC5AC (amino acid residues 1575-2052,Cys2-Cys4). More preferably, the antibodies bind to an epitope locatedin amino acid residues 1575-1725 and 1903-2052 (Cys2 and Cys 4). Evenmore preferably, the antibodies bind to an epitope located in amino acidresidues 1575-1725 (Cys2+). Most preferably, the antibodies bind to anepitope located in Cys2.

In preferred embodiments, the subject antibodies or fragments thereofbind specifically to pancreatic cancer cells, with little or no bindingto normal or non-neoplastic pancreatic cells. The antibodies are capableof binding to the earliest stages of pancreatic cancer, with detectionrates of about 50-60% for PanIN-1A, 70-80% for Pan1B and 80-90% forPanIN-2. More preferably, the antibodies bind to 80 to 90% or more ofhuman invasive pancreatic adenocarcinoma, intraductal papillary mucinousneoplasia, PanIN-1A, PanIN-1B and PanIN-2 lesions. Most preferably, theantibodies can distinguish between early stage pancreatic cancer andnon-malignant conditions such as pancreatitis.

Such antibodies are of particular use for early detection of cancer anddifferential diagnosis between early stage pancreatic cancer and benignpancreatic conditions. In preferred embodiments, such antibodies are ofuse for in vivo or ex vivo analysis of samples from individualssuspected of having early stage pancreatic or certain other cancers.More preferably, the antibodies are of use for detection and diagnosisof early stage pancreatic cancer by analysis of serum samples.

In alternative embodiments, the antibodies, antibody fragments or fusionproteins are capable of binding to synthetic peptide sequences, forexample to phage display peptides, such as WTWNITKAYPLP (SEQ ID NO:7)and ACPEWWGTTC (SEQ ID NO:8). Such synthetic peptides may be linear orcyclic and may or may not compete with antibody binding to theendogenous pancreatic cancer antigen. Amino acids in certain positionsof the synthetic peptide sequences may be less critical for antibodybinding than others. For example, in SEQ ID NO:7 the residues K, A and Lat positions 7, 8 and 11 of the peptide sequence may be varied whilestill retaining antibody binding. Similarly, in SEQ ID NO:8 thethreonine residues at positions 8 and 9 of the sequence may be varied,although substitution of the threonine at position 9 may significantlyaffect antibody binding to the peptide.

Binding of the antibodies to a target pancreatic cancer antigen may beinhibited by treatment of the target antigen with reagents such asdithiothreitol (DTT) and/or periodate. Thus, binding of the antibodiesto a pancreatic cancer antigen may be dependent upon the presence ofdisulfide bonds and/or the glycosylation state of the target antigen. Inmore preferred embodiments, the epitope recognized by the subjectantibodies is not cross-reactive with other reported mucin-specificantibodies, such as the MA5 antibody, the CLH2-2 antibody and/or the45M1 antibody (see, e.g., Major et al., J Histochem Cytochem. 35:139-48,1987; Dion et al., Hybridoma 10:595-610, 1991).

The subject antibodies or fragments may be naked antibodies or fragmentsor preferably are conjugated to at least one therapeutic and/ordiagnostic agent for delivery of the agent to target tissues. Inalternative embodiments, the subject antibodies or fragments may be partof a bispecific antibody with a first binding site for an epitope ofMUC5AC as discussed above and a second binding site for a haptenconjugated to a targetable construct. The targetable construct may inturn be attached to at least one therapeutic and/or diagnostic agent, ofuse in pretargeting techniques.

In preferred embodiments, the subject antibody, antibody fragment orfusion protein is a humanized PAM4 antibody or fragment, comprising thelight chain variable region CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); andthe heavy chain variable region CDR sequences CDR1 (SYVLH, SEQ ID NO:4);CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6)and human antibody framework region (FR) and constant region sequences.More preferably, the FRs of the light and heavy chain variable regionsof the humanized PAM4 antibody or fragment thereof comprise at least oneamino acid substituted from amino acid residues 5, 27, 30, 38, 48, 66,67 and 69 of the murine PAM4 heavy chain variable region (SEQ ID NO: 12)and/or at least one amino acid selected from amino acid residues 21, 47,59, 60, 85, 87 and 100 of the murine PAM4 light chain variable region(SEQ ID NO: 10). Most preferably, the antibody or fragment thereofcomprises the hPAM4 V_(H) amino acid sequence of SEQ ID NO: 19 and thehPAM4 VK amino acid sequence of SEQ ID NO: 16.

In alternative embodiments, the anti-pancreatic cancer antibody may be achimeric, humanized or human antibody that binds to the same antigenicdeterminant (epitope) as, or competes for binding to MUC5AC with, achimeric PAM4 (cPAM4) antibody. As discussed below, the cPAM4 antibodyis one that comprises the light chain variable region CDR sequences CDR1(SASSSVSSSYLY, SEQ ID NO: 1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3(HQWNRYPYT, SEQ ID NO:3); and the heavy chain variable region CDRsequences CDR1 (SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ IDNO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6). Antibodies that bind to thesame antigenic determinant may be identified by a variety of techniquesknown in the art, such as by competitive binding studies using the cPAM4antibody as the competing antibody and human pancreatic mucin or MUC5ACas the target antigen. Antibodies that block (compete for) binding tohuman pancreatic mucin by a cPAM4 antibody are referred to ascross-blocking antibodies. Preferably, such cross-blocking antibodiesare ones that bind to an epitope located within the second to fourthcysteine-rich subdomains of MUC5AC, or that compete for binding to aminoacid residues 1575-2052 with a PAM4 antibody. More preferably, theantibodies bind to an epitope located in Cys2.

Other embodiments concern cancer cell-targeting therapeuticimmunoconjugates comprising an antibody or fragment thereof or fusionprotein bound to at least one therapeutic agent. Preferably, thetherapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an oligonucleotide such as an anti-sense oligonucleotide or asiRNA, an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxicagent such as a drug or toxin, an angiogenesis inhibitor and apro-apoptotic agent. In embodiments where more than one therapeuticagent is used, the therapeutic agents may comprise multiple copies ofthe same therapeutic agent or else combinations of different therapeuticagents. More preferably, the therapeutic agent is a radionuclide, suchas ⁹⁰Y. The labeled antibody may be administered alone, or incombination with one or more other therapeutic agents, such as low-dosegemcitabine.

In certain embodiments, the therapeutic agent is a cytotoxic agent, suchas a drug or a toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicins and theiranalogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, Bruton tyrosinekinase inhibitors, mTOR inhibitors, heat shock protein (HSP90)inhibitors, proteosome inhibitors, HDAC inhibitors, pro-apoptoticagents, methotrexate, CPT-11, SN-38, 2-PDOX, pro-2-PDOX, and acombination thereof.

In another preferred embodiment, the therapeutic agent is a toxinselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin and combinations thereof. Or animmunomodulator selected from the group consisting of a cytokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combinations thereof.

Alternatively, the therapeutic agent is an enzyme selected from thegroup consisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Such enzymes may be used, for example, incombination with prodrugs that are administered in relatively non-toxicform and converted at the target site by the enzyme into a cytotoxicagent. In other alternatives, a drug may be converted into less toxicform by endogenous enzymes in the subject but may be reconverted into acytotoxic form by the therapeutic enzyme.

Other therapeutic agents include radionuclides such as ¹⁴C, ¹³N, ¹⁵O,³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁷Ga, ⁷⁵Br,⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo,^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt, ¹¹¹Ag,¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te, ^(122m)Te, ¹²⁵I, ^(125m)Te, ¹²⁶I,¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁹⁹Au, ²⁰¹Tl,²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po, ²¹⁷At, ²¹⁹Rn,²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²⁵⁵Fm or ²²⁷Th.

A variety of tyrosine kinase inhibitors are known in the art and anysuch known therapeutic agent may be utilized. Exemplary tyrosine kinaseinhibitors include, but are not limited to canertinib, dasatinib,erlotinib, gefitinib, imatinib, lapatinib, leflunomide, nilotinib,pazopanib, semaxinib, sorafenib, sunitinib, sutent and vatalanib. Aspecific class oftyrosine kinase inhibitor is the Bruton tyrosine kinaseinhibitor. Bruton tyrosine kinase (Btk) has a well-defined role inB-cell development. Bruton kinase inhibitors include, but are notlimited to, PCI-32765 (ibrutinib), PCI-45292, GDC-0834, LFM-A13 andRN486.

The subject antibody or fragment may be conjugated to at least onediagnostic (or detection) agent. Preferably, the diagnostic agent isselected from the group consisting of a radionuclide, a contrast agent,a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, aparamagnetic ion, an enzyme and a photoactive diagnostic agent. Stillmore preferred, the diagnostic agent is a radionuclide with an energybetween 20 and 4,000 keV or is a radionuclide selected from the groupconsisting of ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co,⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, orpositron-emitters. In a particularly preferred embodiment, thediagnostic radionuclide ¹⁸F is used for labeling and PET imaging, asdescribed in the Examples below. The ¹⁸F may be attached to an antibody,antibody fragment or peptide by complexation to a metal, such asaluminum, and binding of the ¹⁸F-metal complex to a chelating moietythat is conjugated to a targeting protein, peptide or other molecule.

Also preferred, the diagnostic agent is a paramagnetic ion, such aschromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), or a radiopaque material, such asbarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

In still other embodiments, the diagnostic agent is a fluorescentlabeling compound selected from the group consisting of fluoresceinisothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine, a chemiluminescent labeling compoundselected from the group consisting of luminol, isoluminol, an aromaticacridinium ester, an imidazole, an acridinium salt and an oxalate ester,or a bioluminescent compound selected from the group consisting ofluciferin, luciferase and aequorin. In another embodiment, thediagnostic immunoconjugates are used in intraoperative, endoscopic, orintravascular tumor diagnosis.

Also contemplated are multivalent, multispecific antibodies or fragmentsthereof comprising at least one binding site that binds to an epitope ofMUC5AC as discussed above and one or more hapten binding sites havingaffinity towards hapten molecules. Preferably, the antibody or fragmentthereof is a chimeric, humanized or fully human antibody or fragmentthereof. The hapten molecule may be conjugated to a targetable constructfor delivery of one or more therapeutic and/or diagnostic agents. Incertain preferred embodiments, the multivalent antibodies or fragmentsthereof may be prepared by the DOCK-AND-LOCK™ (DNL™) technique, asdescribed below. An exemplary DNL™ construct incorporating hPAM4antibody fragments is designated TF10, as described below.

Also contemplated is a bispecific antibody or fragment thereofcomprising at least one binding site with an affinity toward an epitopeof MUC5AC as discussed above and at least one binding site with anaffinity toward a targetable construct which is capable of carrying atleast one diagnostic and/or therapeutic agent. Targetable constructssuitable for use are disclosed, for example, in U.S. Pat. Nos.6,576,746; 6,962,702; 7,052,872; 7,138,103; 7,172,751; 7,405,320;7,597,876; 7,563,433; 7,993,626; 8,147,799; 8,153,100; 8,153,101;8,202,509; 8,343,460; 8,444,956, 8,496,912; 8,545,809; 8,617,518; and8,632,752, the Examples section of each of which is incorporated hereinby reference.

Other embodiments concern fusion proteins comprising at least twoanti-pancreatic cancer antibodies and fragments thereof as describedherein. Alternatively, the fusion protein or fragment thereof maycomprise at least one first antibody or fragment thereof that binds toan epitope of MUC5AC as discussed above and at least one second MAb orfragment thereof. Preferably, the second MAb binds to a tumor-associatedantigen, for example selected from the group consisting of CA19.9,DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA (CEACAM5), CEACAM6, Le^(a), theLewis antigen Le(y), CSAp, insulin-like growth factor (IGF), epithelialglycoprotein-1 (EGP-1), epithelial glycoprotein-2 (EGP-2), CD-80,placental growth factor (PlGF), carbonic anhydrase IX, tenascin, IL-6,HLA-DR, CD40, CD74 (e.g., milatuzumab), CD138 (syndecan-1), CTLA-4,PD-1, PD-L1, TIM-3, LAG-3, matrix metalloproteinase-1 (MMP-1), MMP-2,MMP-7, MMP-9, MMP-14, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC16, MUC17,TAG-72, EGFR, platelet-derived growth factor (PDGF), angiogenesisfactors (e.g., VEGF and PlGF), products of oncogenes (e.g., bcl-2, Kras,p53), cMET, HER2/neu, and antigens associated with gastric cancer andcolorectal cancer. The second MAb may also bind to a different epitopeof MUC5AC than the first MAb. The antibody fusion protein or fragmentsthereof may further comprise at least one diagnostic and/or therapeuticagent.

Also described herein are DNA sequences comprising a nucleic acidencoding an anti-pancreatic cancer antibody, fusion protein,multispecific antibody, bispecific antibody or fragment thereof asdescribed herein. Other embodiments concern expression vectors and/orhost cells comprising the antibody-encoding DNA sequences. In certainpreferred embodiments, the host cell may be an Sp2/0 cell linetransformed with a mutant Bcl-2 gene, for example with a triple mutantBcl-2 gene (T69E, S70E, S87E), that has been adapted to celltransformation and growth in serum free medium. (See, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; and 7,608,425, the Examples section of eachof which is incorporated herein by reference.)

Another embodiment concerns methods of delivering a diagnostic ortherapeutic agent, or a combination thereof, to a target comprising (i)providing a composition that comprises an anti-pancreatic cancerantibody or fragment that binds to an epitope located within the secondto fourth cysteine-rich subdomains of MUC5AC (amino acid residues1575-2052), more preferably to an epitope located in amino acid residues1575-1725 and 1903-2052 (Cys2 and Cys 4), even more preferably to anepitope located in amino acid residues 1575-1725 (Cys2+), mostpreferably, to an epitope located in Cys2, conjugated to at least onediagnostic and/or therapeutic agent and (ii) administering to a subjectin need thereof the diagnostic or therapeutic conjugate of any one ofthe antibodies, antibody fragments or fusion proteins claimed herein.

Also contemplated is a method of delivering a diagnostic agent, atherapeutic agent, or a combination thereof to a target, comprising: (a)administering to a subject any one of the multivalent, multispecific orbispecific antibodies or fragments thereof that have an affinity towardan epitope of MUC5AC as discussed above and comprising one or morehapten binding sites; (b) waiting a sufficient amount of time forantibody that does not bind to MUC5AC to clear the subject's bloodstream; and (c) administering to said subject a carrier moleculecomprising a diagnostic agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of the antibody. Preferably, thecarrier molecule binds to more than one binding site of the antibody.

Described herein is a method for diagnosing or treating cancer,comprising: (a) administering to a subject any one of the multivalent,multispecific antibodies or fragments thereof claimed herein that havean affinity toward an epitope of MUC5AC as discussed above andcomprising one or more hapten binding sites; (b) waiting a sufficientamount of time for an amount of the non-bound antibody to clear thesubject's blood stream; and (c) administering to said subject a carriermolecule comprising a diagnostic agent, a therapeutic agent, or acombination thereof, that binds to a binding site of the antibody. In apreferred embodiment the cancer is pancreatic cancer. Also preferred,the method can be used for intraoperative identification of diseasedtissues, endoscopic identification of diseased tissues, or intravascularidentification of diseased tissues.

Another embodiment is a method of treating a malignancy in a subjectcomprising administering to said subject a therapeutically effectiveamount of an antibody or fragment thereof that binds to an epitope ofMUC5AC as discussed above, optionally conjugated to at least onetherapeutic agent, such as ⁹⁰Y. The antibody or fragment thereof mayalternatively be a naked antibody or fragment thereof. In more preferredembodiments, the antibody or fragment is administered either before,simultaneously with, or after administration of another therapeuticagent as described above.

Contemplated herein is a method of diagnosing a malignancy in a subject,particularly a pancreatic cancer, comprising (a) administering to saidsubject a diagnostic conjugate comprising an antibody or fragmentthereof that binds to an epitope of MUC5AC as discussed above, whereinsaid MAb or fragment thereof is conjugated to at least one diagnosticagent, and (b) detecting the presence of labeled antibody bound topancreatic cancer cells or other malignant cells, wherein binding of theantibody is diagnostic for the presence of pancreatic cancer or anothermalignancy. In preferred embodiments, the antibody or fragment binds topancreatic cancer and not to normal pancreatic tissue, pancreatitis orother non-malignant conditions. In less preferred embodiments, theantibody or fragment binds at a significantly higher level to cancercells than to non-malignant cells, allowing differential diagnosis ofcancer from non-malignant conditions. In a most preferred embodiment,the diagnostic agent may be an F-18 labeled molecule that is detected byPET imaging.

In more preferred embodiments, the use of anti-pancreatic cancerantibodies that bind to an epitope located within the second to fourthcysteine-rich subdomains of MUC5AC (amino acid residues 1575-2052), morepreferably to an epitope located in amino acid residues 1575-1725 and1903-2052 (Cys2 and Cys 4), even more preferably to an epitope locatedin amino acid residues 1575-1725 (Cys2+), most preferably, to an epitopelocated in Cys2, allows the detection and/or diagnosis of pancreaticcancer with high specificity and sensitivity at the earliest stages ofmalignant disease. Preferably, the diagnostic antibody or fragment iscapable of labeling at least 70%, more preferably at least 80%, morepreferably at least 90%, more preferably at least 95%, most preferablyabout 100% of well differentiated, moderately differentiated and poorlydifferentiated pancreatic cancer and 90% or more of invasive pancreaticadenocarcinomas. The anti-pancreatic cancer antibody of use ispreferably capable of detecting 85% or more of PanIN-1A, PanIN-1B,PanIN-2, IPMN and MCN precursor lesions. Most preferably, immunoassaysusing the anti-pancreatic cancer antibody are capable of detecting 89%or more of total PanIN, 86% or more of IPMN, and 92% or more of MCN.

An alternative embodiment is a method of detecting the presence ofPAM4-binding MUC5AC and/or diagnosing pancreatic cancer in an individualby in vitro analysis of blood, plasma or serum samples. Preferably, thesample is subjected to an organic phase extraction, using an organicsolvent such as butanol, before it is processed for immunodetectionusing an anti-pancreatic cancer antibody, such as a PAM4 antibody.Following organic phase extraction, the extracted aqueous phase isanalyzed for the presence of the epitope of MUC5AC to which PAM4 bindsin the sample, using any of a variety of immunoassay techniques known inthe art, such as ELISA, sandwich immunoassay, solid phase RIA, andsimilar techniques. Surprisingly, the organic phase extraction resultsin the removal of an inhibitor of PAM4 binding to MUC5AC, allowingdetection of MUC5AC in fresh serum samples. More surprisingly, using thein vitro analysis techniques described herein, serum samples may beanalyzed to detect and/or diagnose pancreatic cancer in a subject at theearliest stages of pancreatic adenocarcinoma. These unexpected resultsprovide the first serum-based assay technique that is diagnostic for thepresence of early stage pancreatic cancer.

Another embodiment is a method of treating a cancer cell in a subjectcomprising administering to said subject a composition comprising anaked antibody or fragment thereof or a naked antibody fusion protein orfragment thereof that binds to an epitope of MUC5AC as discussed above.Preferably, the method further comprises administering a second nakedantibody or fragment thereof selected from the group consisting ofCA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6,anti-EGP-1, anti-EGP-2, anti-Le^(a), antibodies defined by the Lewisantigen Le(y), and antibodies against CSAp, MUC1, MUC2, MUC3, MUC4,MUC5AC, MUC16, MUC17, TAG-72, EGFR, CD40, HLA-DR, CD74, CD138,angiogenesis factors (e.g., VEGF and placenta-like growth factor (PlGF),insulin-like growth factor (IGF), tenascin, platelet-derived growthfactor, IL-6, products of oncogenes, cMET, and HER2/neu.

Still other embodiments concern a method of diagnosing a malignancy in asubject comprising (i) performing an in vitro diagnosis assay on aspecimen from said subject with a composition comprising an antibody orfragment thereof that binds to an epitope of MUC5AC as discussed above;and (ii) detecting the presence of antibody or fragment bound tomalignant cells in the specimen. Preferably, the malignancy is a cancer.More preferably, the cancer is pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate preferred embodimentsof the invention. However, the claimed subject matter is in no waylimited by the illustrative embodiments disclosed in the drawings.

FIG. 1. Co-localization of PAM4 antigen with MUC5AC by cytoflurometricstaining. Mucin-expressing cell lines (Capan-1, BxPC-3, HT-29, andMCF-7) were stained with DAPI, 2-11M1 (anti-MUC5AC), hPAM4, and examinedby immunofluorescence microcopy. BxPC-3 and HT-29 cells were alsostained with anti-MUC1. PAM4 antigen was shown to co-localize withMUC5AC, not MUC1.

FIG. 2. Co-knockdown of PAM4 antigen and MUC5AC by MUC5AC-specificsiRNA. CFPAC-1 cells were treated with a MUC5AC-specific siRNA, followedby immunostaining with DAPI, hPAM4, and 2-11M1 (anti-MUC5AC), or withDAPI, hPAM4 and anti-MUC1. Untreated Cells or cells treated with onlythe transfection agent (Mock) served as controls. Cells treated withMUC5AC-specific siRNA lost the binding to anti-MUC5AC and hPAM4concurrently, with little effect on the binding to anti-MUC1.

FIG. 3A. Immunoreactivity of fractions eluted from SEPHAROSE® CL-2B.Capan-1 cell culture supernatant was separated on a SEPHAROSE® CL2Bcolumn with the eluted fractions analyzed by hPAM4 and anti-MUC1.

FIG. 3B. Immunoreactivity of fractions eluted from SEPHAROSE® CL-2B. Thevoid-volume (Vo) fractions of Capan-1 reacted positively with threeanti-MUC5AC antibodies (45M1, 1-13M1 and H-160), but not with 2Q445,which recognizes the unglycosylated tandem repeat region of MUC5AC.

FIG. 3C. Immunoreactivity of fractions eluted from SEPHAROSE® CL-2B. TheCapan-1 void-volume peak, following capture by 2-11M1, could be detecteddirectly by HRP-hPAM4, or indirectly by biotin-45M1 plus SA-HRP.

FIG. 4A. Agarose gel electrophoresis. The Capan-1 void-volume peakdisplayed the characteristic banding pattern of MUC5AC as revealed byWestern blot analysis with hPAM4, 45M1, and MAN-5ACI. In the left panel,samples in the lanes marked as 1, ½, ¼, and ⅛ were tested undiluted, 2-,4- and 8-fold diluted, respectively. In the far right panel, themonomeric and dimeric MUC5AC were indicated as M and D, respectively.

FIG. 4B. Agarose gel electrophoresis. The serum from a pancreatic cancerpatient (PS) tested positive for hPAM4-reactive substance wasdifferentially detected by hPAM4 and three anti-MUC5AC antibodies(2-11M1, 45M1, and H-160). The Capan-1 void-volume peak (Vo) and normalserum sample (NS) were included as controls.

FIG. 5A. Mapping the PAM4-reactive epitope on human MUC5AC. Schematicdiagram of different MUC5AC recombinant fragments (a-h) generated inPANC-1 cells for mapping PAM4 epitope; Numbers are AA positions in theMUC5AC protein sequence (UniProtKB/Swiss-Prot: P98088).

FIG. 5B. Mapping the PAM4-reactive epitope on human MUC5AC. Western blotof a-fragment (AA3992-5030), b-fragment (AA1-1218) and c-fragment(AA1218-2199), which spans the five N-terminal cysteine-rich subdomains(Cys1-2-3-4-5), with hPAM4 and 45M1 antibodies. Lane m indicates samplesfrom untransfected cells.

FIG. 5C. Mapping the PAM4-reactive epitope on human MUC5AC. Western blotof c-fragment (AA1218-2199), d-fragment (AA1218-1517) and e-fragment(AA1575-2052), with hPAM4, H160 and 45M1 antibodies. Lane m indicatessamples from untransfected cells.

FIG. 5D. Mapping the PAM4-reactive epitope on human MUC5AC. Western blotof e-fragment (AA1575-2052), f-fragment (AA1725-2052), g-fragment(AA1575-1725 and 1903-2052) and h-fragment (AA1575-1853) with hPAM4 and45M1 antibodies. Lane m indicates samples from untransfected cells.

FIG. 5E. Mapping the PAM4-reactive epitope on human MUC5AC. Western blotof GFP-fused e*-fragment (AA1575-2052), f*-fragment (AA1725-2052),g*-fragment (AA1575-1725 and 1903-2052) and h*-fragment (AA1575-1853)with hPAM4 and anti-GFP antibodies. Lane m indicates samples fromuntransfected cells.

FIG. 6A. SDS-PAGE and Western blot analyses of recombinant MUC5ACfragments expressed in E. coli. Four gels were run under similarconditions of SDS-PAGE. Gel was stained with coomassie blue. Samples,either reduced (R) or non-reduced (NR), were loaded at 500 ng/well; laneM, markers; lanes 1 & 3, Cys2-3-4 (AA1575-2052); lanes 2 &4,Cys2+(AA1575-1725).

FIG. 6B. SDS-PAGE and Western blot analyses of recombinant MUC5ACfragments expressed in E. coli. Four gels were run under similarconditions of SDS-PAGE. Gel was transferred onto nitrocellulose membraneand stained with anti-Myc. Samples, either reduced (R) or non-reduced(NR), were loaded at 500 ng/well; lane M, markers; lanes 1 & 3, Cys2-3-4(AA1575-2052); lanes 2 &4, Cys2+(AA1575-1725).

FIG. 6C. SDS-PAGE and Western blot analyses of recombinant MUC5ACfragments expressed in E. coli. Four gels were run under similarconditions of SDS-PAGE. Gel was transferred onto nitrocellulose membraneand stained with hPAM4. Samples, either reduced (R) or non-reduced (NR),were loaded at 500 ng/well; lane M, markers; lanes 1 & 3, Cys2-3-4(AA1575-2052); lanes 2 &4, Cys2+(AA1575-1725).

FIG. 6D. SDS-PAGE and Western blot analyses of recombinant MUC5ACfragments expressed in E. coli. Four gels were run under similarconditions of SDS-PAGE. Gel was transferred onto nitrocellulose membraneand stained with 1-13M1. Samples, either reduced (R) or non-reduced(NR), were loaded at 500 ng/well; lane M, markers; lanes 1 & 3, Cys2-3-4(AA1575-2052); lanes 2 &4, Cys2+(AA1575-1725).

FIG. 7. Overall survival. Kaplan-Meier curves and time-point analysesfor all 29 patients in Arm A (⁹⁰Y-clivatuzumab tetraxetan combined withlow-dose gemcitabine) and all 29 patients in Arm B (⁹⁰Y-clivatuzumabtetraxetan alone).

FIG. 8A. Variable region cDNA sequences (SEQ ID NO:9) and the deducedamino acid sequences (SEQ ID NO: 10) of the murine PAM4 Vk. Amino acidsequences encoded by the corresponding DNA sequences are given asone-letter codes below the nucleotide sequence. Numbering of thenucleotide sequence is on the right side. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering is used for amino acid residues as shown by the numberingabove the amino acid residues. The amino acid residues numbered by aletter are the insertion residues defined by Kabat numbering scheme. Theinsertion residues have the same preceding digits as that of theprevious residue.

FIG. 8B. Variable region cDNA sequence (SEQ ID NO: 11) and the deducedamino acid sequence (SEQ ID NO: 12) of the murine PAM4 VH. Amino acidsequences encoded by the corresponding DNA sequences are given asone-letter codes below the nucleotide sequence. Numbering of thenucleotide sequence is on the right side. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering is used for amino acid residues as shown by the numberingabove the amino acid residues. The amino acid residues numbered by aletter are the insertion residues defined by Kabat numbering scheme. Theinsertion residues have the same preceding digits as that of theprevious residue.

FIG. 9A. Amino acid sequence (SEQ ID NO: 13) of the chimeric PAM4(cPAM4) Vk. The sequences are given as one letter codes. The amino acidresidues in the CDR regions are shown in bold and underlined. Kabat's Igmolecule number scheme is used to number the residues.

FIG. 9B. Amino acid sequence (SEQ ID NO: 14) of the cPAM4 VH. Thesequences are given as one letter codes. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig molecule numberscheme is used to number the residues.

FIG. 10A. Alignment of the VK amino acid sequences of the human antibodyWalker (SEQ ID NO:15) with PAM4 (SEQ ID NO:10) and hPAM4 (SEQ ID NO:16).Dots indicate the residues of PAM4 that are identical to thecorresponding residues of the human or humanized antibodies. Boxedregions represent the CDR regions. Both N- and C-terminal residues(underlined) of hPAM4 are fixed by the staging vectors used. Kabat's Igmolecule number scheme is used to number the residues.

FIG. 10B. Alignment of the VH amino acid sequences of the human antibodyWil2 (FR1-3) (SEQ ID NO:17) and NEWM (FR4) (SEQ ID NO:18) with PAM4 (SEQID NO:12) and hPAM4 (SEQ ID NO: 19). Dots indicate the residues of PAM4that are identical to the corresponding residues of the human orhumanized antibodies. Boxed regions represent the CDR regions. Both N-and C-terminal residues (underlined) of hPAM4 are fixed by the stagingvectors used. Kabat's Ig molecule number scheme is used to number theresidues.

FIG. 11A. DNA (SEQ ID NO:20) and amino acid (SEQ ID NO:16) sequences ofthe humanized PAM4 (hPAM4) Vk. Numbering of the nucleotide sequence ison the right side. Amino acid sequences encoded by the corresponding DNAsequences are given as one-letter codes. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering scheme is used for amino acid residues.

FIG. 11B. DNA (SEQ ID NO:21) and amino acid (SEQ ID NO: 19) sequences ofthe hPAM4 VH. Numbering of the nucleotide sequence is on the right side.Amino acid sequences encoded by the corresponding DNA sequences aregiven as one-letter codes. The amino acid residues in the CDR regionsare shown in bold and underlined. Kabat's Ig molecule numbering schemeis used for amino acid residues.

FIG. 12. Binding activity of humanized PAM4 antibody, hPAM4, as comparedto the chimeric PAM4, cPAM4. hPAM4 is shown by diamonds and cPAM4 isshown by closed circles. Results indicate comparable binding activity ofthe hPAM4 antibody and cPAM4 when competing with ¹²⁵I-cPAM4 binding toCaPan1 antigens.

FIG. 13. PET/CT fusion images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). The circleindicates the location of the primary lesion, which shows a significantdecrease in PET/CT intensity following therapy.

FIG. 14. 3D PET images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). Arrows pointto the locations of the primary lesion (on right) and metastases (onleft), each of which shows a significant decrease in PET image intensityafter therapy with radiolabeled hPAM4 plus gemcitabine.

FIG. 15A. In vivo imaging of tumors using an ¹¹¹In-labeled diHSG peptide(IMP 288) with or without pretargeting TF10 bispecific anti-pancreaticcancer MUC5AC antibody. FIG. 15A illustrates mice showing the locationof tumors (arrow).

FIG. 15B In vivo imaging of tumors using an ¹¹¹In-labeled diHSG peptide(IMP 288) with or without pretargeting TF10 bispecific anti-pancreaticcancer MUC5AC antibody. FIG. 15B shows the detected tumors with¹¹¹In-labeled IMP 288 in the presence (above) or absence (below) of TF10bispecific antibody.

FIG. 16. Exemplary binding curves for TF10, PAM4-IgG, PAM4-F(ab′)₂ and amonovalent bsPAM4 chemical conjugate (PAM4-Fab′×anti-DTPA-Fab′). Bindingto target mucin antigen was measured by ELISA assay.

FIG. 17A. Immunoscintigraphy of CaPan1 human pancreatic cancerxenografts (˜0.25 g). An image of mice that were injected withbispecific TF10 (80 Gig, 5.07×10⁻¹⁰ mol) followed 16 h later byadministration of ¹¹¹In-IMP-288 (30 μCi, 5.07×10⁻¹¹ mol). The image wastaken 3 h later. The intensity of the image background was increased tomatch the intensity of the image obtained when ¹¹¹In-IMP-288 wasadministered alone (30 μCi, 5.07×10⁻¹¹ mol).

FIG. 17B. Immunoscintigraphy of CaPan1 human pancreatic cancerxenografts (˜0.25 g). No targeting was observed in mice given¹¹¹In-IMP-288 alone.

FIG. 17C. Immunoscintigraphy of CaPan1 human pancreatic cancerxenografts (˜0.25 g). An image of mice that were given¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50 μg) with imaging done 24 h later.Although tumors are visible, considerable background activity is stillpresent at this time point.

FIG. 18A. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10followed 16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nudemice bearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 18A shows percent of initial doseper gram of tissue in tumor with PAM4 IgG (open circles), blood withPAM4 IgG (open squares), tumor with pretargeted peptide (closed circles)and blood with pretargeted peptide (closed squares).

FIG. 18B. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10followed 16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nudemice bearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 18B shows percent of initial doseper gram of tissue in liver with PAM4 IgG (open triangles), kidney withPAM4 IgG (open diamonds), liver with pretargeted peptide (closedtriangles) and kidney with pretargeted peptide (closed diamonds).

FIG. 18C. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10followed 16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nudemice bearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 18C shows microcuries per gram oftissue in tumor with PAM4 IgG (open circles), blood with PAM4 IgG (opensquares), tumor with pretargeted peptide (closed circles) and blood withpretargeted peptide (closed squares).

FIG. 18D. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07⁻¹⁰ mol TF10 followed16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nude micebearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 18D shows microcuries per gram oftissue in liver with PAM4 IgG (open triangles), kidney with PAM4 IgG(open diamonds), liver with pretargeted peptide (closed triangles) andkidney with pretargeted peptide (closed diamonds).

FIG. 19. Therapeutic activity of a single treatment of established (˜0.4cm³) CaPan1 tumors with 0.15 mCi of ⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCiof TF10-pretargeted ⁹⁰Y-IMP-288.

FIG. 20. Effect of gemcitabine potentiation of PT-RAIT therapy.

FIG. 21. Effect of combination of cetuximab with gemcitabine andPT-RAIT.

FIG. 22. Differential diagnosis of pancreatic cancer using PAM4-basedimmunoassay. The horizontal line shows the cutoff level selected for apositive result, based on ROC analysis.

FIG. 23. Frequency distribution of PAM4 antigen in patient sera fromhealthy volunteers and individuals with varying stages of pancreaticcancer.

FIG. 24. ROC curve for PAM4 serum immunoassay, showing sensitivity fordetection of 81.6% and specificity of 84.6%.

FIG. 25. Accuracy of the PAM4-immunoassay was determined to be within10% of the nominal concentrations examined at or above the cutoff valueof 2.40 units/mL. A linear trend was calculated with an equation ofy=0.965x+0.174, and goodness of fit r²=0.999.

FIG. 26. Frequency distribution of PAM4-reactive antigen in patient seraby stage of disease. Cutoff value=2.4 units/mL (horizontal line). Themedian values (units/mL) are shown for each study group.

FIG. 27. Receiver Operator Characteristics (ROC) curve for theperformance of the PAM4-based immunoassay; pancreatic adenocarcinoma vs.healthy adults. Values for the area under the curves (AUC) and 95%confidence limits are provided.

FIG. 28A. Circulating PAM4 antigen levels correlated withprogression/regression of tumor volume (CT) following treatment with⁹⁰Y-PAM4-IgG plus gemcitabine. Patient 076-001 was responsive to therapyand serum PAM4 antigen decreased. Serum PAM4 levels correlated withtumor volume.

FIG. 28B. Circulating PAM4 antigen levels correlated withprogression/regression of tumor volume (CT) following treatment with⁹⁰Y-PAM4-IgG plus gemcitabine. Patient 1810002 showed an initialresponse to therapy, followed by recurrence of the tumor. Serum PAM4levels correlated with tumor volume.

FIG. 29. Reactivity of PAM4 with mucin standards in the presence orabsence of palmitic acid.

FIG. 30A. Sensitivity and specificity for PAM4 detection of PDAC vs.chronic pancreatitis (CP).

FIG. 30B. Sensitivity and specificity for PAM4 detection of PDAC vs. allbenign tissue samples.

FIG. 31. Comparative labeling of PDAC vs. non-neoplastic prostate tissuewith PAM4 vs. antibodies against MUC1, MUC4, CEACAM6 and CA19-9.

FIG. 32. Reactivity of several anti-mucin MAbs with a high molecularweight mucin containing fraction (CPM1) isolated from the Capan-1, humanpancreatic adenocarcinoma. MAbs are identified by clone name withreactive species of mucin indicated by horizontal bars beneath MAb clonenames. In addition to PAM4, substantial reactions were observed foranti-MUC1, MUC5AC, and CEACAM6 antibodies. All MAbs were employed at aconstant 10 μg/mL.

FIG. 33. Reaction of several anti-mucin MAbs with PAM4-captured antigen.Mucin antigens were captured on hPAM4 coated plates, and then probedwith several murine anti-mucin MAbs for reaction signal. Bothanti-MUC5AC MAbs (2-11M1 and 45M1) bound to the hPAM4-captured mucin,whereas the anti-MUC1 MAbs (MA5 and KC4) did not bind. The homologoushPAM4/mPAM4, capture/probe immunoassay gave no signal, suggesting thedensity of PAM4 epitopes within the mucin may be low, possibly only asingle site. A rabbit polyclonal anti-CPM1, IgG, was used as a positivecontrol for reaction with hPAM4-captured antigen.

FIG. 34A. Inhibition of hPAM4/antigen binding reaction by murineanti-mucin MAbs. Anti-mucin mMAbs (purified IgG) were added toCPM1-coated plates as potential inhibitors prior to addition of hPAM4.mPAM4 provided almost complete inhibition of the reaction between hPAM4and antigen with the 45M1 anti-MUC5AC providing limited inhibitoryaffect (IC_(max)=25.5%). Neither 2-11M1, anti-MUC5AC nor MA5 and KC4,anti-MUC1 MAbs were able to inhibit the specific hPAM4/antigen reaction.

FIG. 34B. Inhibition of hPAM4/antigen binding reaction by murineanti-mucin MAbs. A similar inhibition study was performed with severalanti-MUC5AC MAbs obtained as ascites fluids. MAbs 21M1, 62M1, and 463M1,anti-MUC5AC provided substantial inhibitory affect similar to thatobserved with mPAM4, IgG, self-inhibition. The ascites form of 45M1yielded an inhibitory affect similar to that of the purified IgG.Ascites containing anti-alpha fetoprotein was employed as a negativecontrol.

FIG. 35. Representation of the domains of the MUC5AC glycoprotein withreactive epitopes indicated for several anti-MUC5AC MAbs. Data derivedby transfection with plasmid vectors containing the cDNA of the 3′-endof MUC5AC, along with derivative cDNA vectors obtained by restrictionenzyme digestion, have identified the location of specific epitopes foranti-MUC5AC MAbs employed in the current studies. Specific blockingstudies suggest the PAM4-epitope resides within the cysteine-richC-terminus domain.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

As described herein, the term “PAM4 antibody” includes murine, chimeric,humanized and human PAM4 antibodies. In preferred embodiments, the PAM4antibody or antigen-binding fragment thereof comprises the CDR sequencesof SEQ ID NO: 1 to SEQ ID NO:6.

As used herein, an “anti-pancreatic cancer antibody” is an antibody thatexhibits the same diagnostic, therapeutic and binding characteristics asthe PAM4 antibody. In preferred embodiments, the “anti-pancreatic cancerantibody” binds to an epitope located within the second to fourthcysteine-rich subdomains of MUC5AC (amino acid residues 1575-2052), morepreferably to an epitope located in amino acid residues 1575-1725 and1903-2052 (Cys2 and Cys 4), even more preferably to an epitope locatedin amino acid residues 1575-1725 (Cys2+), most preferably, to an epitopelocated in Cys2.

A “non-endocrine pancreatic cancer” generally refers to cancers arisingfrom the exocrine pancreatic glands. The term excludes pancreaticinsulinomas and includes pancreatic carcinoma, pancreaticadenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma andgiant cell carcinoma and precursor lesions such as pancreaticintra-epithelial neoplasia (PanIN), mucinous cyst neoplasms (MCN) andintrapancreatic mucinous neoplasms (IPMN), which are neoplastic but notyet malignant. The terms “pancreatic cancer” and “non-endocrinepancreatic cancer” are used interchangeably herein.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the full-lengthantibody. The term “antibody fragment” also includes isolated fragmentsconsisting of the variable regions of antibodies, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”). Another form of antibody fragment is a single domainantibody (nanobody).

A naked antibody is an antibody or fragment thereof that is notconjugated to a therapeutic or diagnostic agent. Generally, the Fcportion of the antibody molecule provides effector functions, such ascomplement-mediated cytotoxicity (CDC) and ADCC (antibody-dependentcellular cytotoxicity), which set mechanisms into action that may resultin cell lysis. However, the Fc portion may not be required fortherapeutic function, with other mechanisms, such as signaling-inducedapoptosis, coming into play. Naked antibodies include both polyclonaland monoclonal antibodies, as well as fusion proteins and certainrecombinant antibodies, such as chimeric, humanized or human antibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples sections of which are incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,cytotoxic agents, drugs, toxins, nucleases, hormones, immunomodulators,pro-apoptotic agents, anti-angiogenic agents, boron compounds,photoactive agents or dyes and radioisotopes. Therapeutic agents of useare described in more detail below.

A diagnostic agent is a molecule, atom or other detectable moiety whichmay be administered conjugated to an antibody moiety or targetableconstruct and is useful in detecting or diagnosing a disease by locatingcells containing the target antigen. Useful diagnostic agents include,but are not limited to, radioisotopes, dyes, contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.,paramagnetic ions) for magnetic resonance imaging (MRI) or positronemission tomography (PET) scanning. Preferably, the diagnostic agentsare selected from the group consisting of radioisotopes, enhancingagents for use in magnetic resonance or PET imaging, and fluorescentcompounds. In order to load an antibody component with radioactivemetals, paramagnetic ions or other diagnostic cations, it may benecessary to react it with a reagent having a long tail to which areattached a multiplicity of chelating groups for binding the ions. Such atail can be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chain having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA,porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,polyoximes, and like groups known to be useful for this purpose.Chelates are coupled to the antibodies using standard chemistries. Thechelate is normally linked to the antibody by a group which enablesformation of a bond to the molecule with minimal loss ofimmunoreactivity and minimal aggregation and/or internal cross-linking.Other, more unusual, methods and reagents for conjugating chelates toantibodies are disclosed in U.S. Pat. No. 4,824,659, the Examplessection of which is incorporated herein by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I,⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O,⁷⁶Br, for radioimaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along with the antibodies of the invention.Macrocyclic chelates such as NOTA(1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA(1,4,7,10-tetraazacyclododecanetetraacetic acid), and TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of usewith a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for radioimmunotherapy (RAIT) are encompassed bythe invention. More recently, techniques of general utility for labelingvirtually any molecule with an ¹⁸F atom of use in PET imaging have beendescribed in U.S. Pat. Nos. 7,563,433; 7,597,876 and 7,993,626, theExamples section of each incorporated herein by reference.

An immunoconjugate is an antibody, antibody fragment or antibody fusionprotein conjugated to at least one therapeutic and/or diagnostic agent.The diagnostic agent and/or therapeutic agent are as defined above.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as transgenicanimals, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell. Suitable mammalianhost cells include myeloma cells, such as SP2/0 cells, and NS0 cells, aswell as Chinese Hamster Ovary (CHO) cells, hybridoma cell lines andother mammalian host cell useful for expressing antibodies. Alsoparticularly useful to express mAbs and other fusion proteins are Sp2/0cells transfected with an apoptosis inhibitor, such as a Bcl-EEE gene,and adapted to grow and be further transfected in serum free conditions,as described in U.S. Pat. Nos. 7,531,327; 7,537,930; and 7,608,425, theExamples section of each of which is incorporated herein by reference.

Anti-Pancreatic Cancer Antibodies

Various embodiments of the invention concern antibodies that react withvery high selectivity with pancreatic cancer as opposed to normal orbenign pancreatic tissues. The anti-pancreatic cancer antibodies andfragments thereof are preferably raised against a crude mucinpreparation from a tumor of the human pancreas, although partiallypurified or even purified MUC5AC may be utilized. A non-limiting exampleof such antibodies is the PAM4 antibody.

The murine PAM4 (mPAM4) antibody was developed by employing pancreaticcancer mucin derived from the xenografted RIP-1 human pancreaticcarcinoma as immunogen. (Gold et al., Int. J. Cancer, 57:204-210, 1994.)As discussed below, antibody cross-reactivity and immunohistochemicalstaining studies indicate that the PAM4 antibody recognizes a unique andnovel epitope on MUC5AC. Immunohistochemical staining studies have shownthat the PAM4 MAb binds to an antigen expressed by breast, pancreas andother cancer cells, with limited binding to normal human tissue.However, the highest expression is usually by pancreatic cancer cells.Thus, the PAM4 antibodies are relatively specific to pancreatic cancerand preferentially bind pancreatic cancer cells. The PAM4 antibody isreactive with a target epitope which can be internalized. This epitopeis expressed primarily by antigens associated with pancreatic cancer andnot with focal pancreatitis or normal pancreatic tissue. Binding of PAM4antibody to the PAM4 epitope is inhibited by treatment of the antigenwith DTT or periodate. Localization and therapy studies using aradiolabeled PAM4 MAb in animal models have demonstrated tumor targetingand therapeutic efficacy.

The PAM4 antibodies bind to an epitope located within the second tofourth cysteine-rich subdomains of MUC5AC (amino acid residues1575-2052), more preferably to an epitope located in amino acid residues1575-1725 and 1903-2052 (Cys2 and Cys 4), even more preferably to anepitope located in amino acid residues 1575-1725 (Cys2+), mostpreferably, to an epitope located in Cys2. The PAM4 epitope is expressedby many organs and tumor types, but is preferentially expressed inpancreatic cancer cells. Studies with a PAM4 MAb, as in the Examplesbelow, indicate that the antibody exhibits several important properties,which make it a good candidate for clinical diagnostic and therapeuticapplications. The epitope provides a useful target for diagnosis andtherapy of pancreatic and other cancers. The PAM4 antibody apparentlyrecognizes an epitope of MUC5AC that is distinct from the epitopesrecognized by non-PAM4 anti-pancreatic cancer antibodies (e.g., CA19.9,DUPAN2, SPAN1, Nd2, CEACAM5, B72.3, anti-Le^(a), and other anti-Lewisantigens).

Surprisingly, the Examples below indicate that the MUC5AC epitope towhich PAM4 binds is present in detectable concentrations in serum ofpatients with very early stage pancreatic cancer. Also surprisingly, itappears that an endogenous inhibitor of PAM4 antibody binding to MUC5ACis present in fresh human serum. The inhibitor is removed by long-termfrozen storage of serum samples, or by organic phase extraction of freshserum. These unexpected results provide the basis of a relativelynon-invasive, early detection test for pancreatic cancer, using blood,serum or plasma samples. In alternative embodiments, the PAM4 antibodymay be used alone, or else in conjunction with one or more otherantibodies, such as CA19.9 antibody, to detect pancreatic cancer markersin serum.

At the tissue level, the reactivity of PAM4 is highly restricted toPDAC, with the biomarker expressed (or becomes accessible) at theearliest stages of neoplastic development (Gold et al., 1994, Int JCancer 57:204-10; Gold et al., 2007, Clin Cancer Res 13:7380-7),including pancreatic intraepithelial neoplasia (PanIN), and intraductalpapillary mucinous neoplasm (IPMN). Notably, the PAM4-biomarker isabsent from normal pancreas and benign, non-neoplastic lesions. In over50 surgical specimens of chronic pancreatitis, the PAM4-biomarker wasidentified only within associated PanIN lesions and not by the inflamedparenchyma, including ducts, acinar cells, and acinar-ductal metaplasia(Shi et al., 2014, Arch Pathol Lab Med 138:220-8).

Preclinical studies have demonstrated the potential applications of PAM4for radioimmunoimaging and radioimmunotherapy of pancreatic carcinoma(Gold et al., 2002, Crit Rev Oncol Hematol 39:147-54; Gold et al., 2004,Int J Cancer 109: 618-26). In patients, ⁹⁰Y-labeled, humanized PAM4(⁹⁰Y-clivatuzumab tetraxetan, hereafter referred to as ⁹⁰Y-hPAM4) waswell tolerated with manageable hematologic toxicity under maximaltolerated ⁹⁰Y dosing, and produced objective responses in bothchemotherapy-naïve and -refractory patients with advanced PDAC (Gulec etal., 2011, Clin Cancer Res 17:4091-100). Further, ⁹⁰Y-hPAM4 incombination with low-dose gemcitabine showed enhanced therapeuticefficacy in patients with metastatic pancreatic cancer (Ocean et al.,2012, Cancer 118:5497-506). In a recently completed phase Ib study(Picozzi et al., 2014, J Clin Oncol 132:4026) involving 58 patients withmetastatic PDAC who had at least 2 prior therapies, multiple cycles offractionated ⁹⁰Y-hPAM4 in combination with low radiosensitizing doses ofgemcitabine significantly (P=0.004) improved the Kaplan-Meier medianoverall survival of this difficult-to-treat population to 7.9 months,compared to those receiving only ⁹⁰Y-hPAM4 (3.4 months). These promisingresults led to the ongoing phase III registration trial of ⁹⁰Y-hPAM4 incombination with gemcitabine (NCT01956812).

In addition, PAM4 or hPAM4-based ELISA has been devised and evaluatedfor detection of PDAC, showing that nearly two-thirds of patients havingconfirmed stage-1 disease had elevated PAM4 antigen in their serum (Goldet al., 2010, Cancer Epidemiol Biomarkers Prev 19:2786-94). However, thecurrent assay, which employs hPAM4 as the capture antibody and apolyclonal rabbit anti-mucin antiserum (IgG fraction) as a probe, is notoptimal, because the polyclonal probe is available in only limitedquantities and, more importantly, is not itself specific for the PAM4antigen. Another concern for further development of the assay has beenthe unknown nature of the antigen marker to which PAM4 is reactive.Given the clinical merit and ongoing evaluation of hPAM4 as a potentialdiagnostic and therapeutic agent for PDAC, there is an urgent need toidentify the PAM4 epitope. Towards this end, we recently proposed (Goldet al., 2013, Mol Cancer 12:143) that PAM4 was reactive with the humanMUC5AC, a polymeric gel-forming mucin with the monomeric form consistingof more than 5,000 amino acid residues organized into three majorregions (Thornton et al., 2008, Annu Rev Physiol 70:459-86): a signalpeptide and four von Willebrand factor (vWF)-like cysteine-rich domains(D1, D2, D′ and D3) in the N-terminal region, a MUC11p15-type domainpreceding the heavily O-glycosylated mucin domain in the central region,and a cluster of vWF-like cysteine-rich domains (D4, B, C, and CK) inthe C-terminal region. In addition, 9 cysteine-rich subdomains(designated Cys1, Cys2, Cys3, Cys4, Cys5, Cys6, Cys7, Cys8, and Cys9)are interspersed within the mucin domain. Herein we present furtherevidence to support MUC5AC as the PAM4-reactive mucin and, importantly,have mapped the PAM4 epitope to Cys2.

For therapeutic use, antibodies suitable for use in combination orconjunction with PAM4 antibodies include, for example, the antibodiesCA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6,anti-Le^(a), anti-HLA-DR, anti-CD40, anti-CD74, anti-CD138, andantibodies defined by the Lewis antigen Le(y), or antibodies againstcolon-specific antigen-p (CSAp), CTLA-4, PD-1, PD-L1, TIM-3, LAG-3,matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-7, MMP-9, MMP-14, MUC1,MUC2, MUC3, MUC4, MUC5AC, MUC16, MUC17, EGP-1, EGP-2, HER2/neu, EGFR,angiogenesis factors (e.g., VEGF and PlGF), insulin-like growth factor(IGF), tenascin, platelet-derived growth factor, and IL-6, as well asproducts of oncogenes (bcl-2, Kras, p53), cMET, and antibodies againsttumor necrosis substances, such as described in patents by Epstein etal. (U.S. Pat. Nos. 6,071,491, 6,017,514, 5,019,368 and 5,882,626). Suchantibodies would be useful for complementing PAM4 antibodyimmunodetection and immunotherapy methods. These and other therapeuticagents could act synergistically with anti-pancreatic cancer antibodies,such as PAM4 antibody, when administered before, together with or afteradministration of PAM4 antibody.

In therapeutic applications, antibodies that are agonistic orantagonistic to immunomodulators involved in effector cell functionagainst tumor cells could also be useful in combination with PAM4antibodies alone or in combination with other tumor-associatedantibodies, one example being antibodies against CD40. Todryk et al., J.Immunol Methods, 248:139-147 (2001); Turner et al., J. Immunol,166:89-94 (2001). Also of use are antibodies against markers or productsof oncogenes (e.g., bcl-2, Kras, p53, cMET), or antibodies againstangiogenesis factors, such as VEGFR and placenta-like growth factor(PlGF).

The availability of another PAM4-like antibody that binds to a differentepitope of MUC5AC is important for the development of adouble-determinant enzyme-linked immunosorbent assay (ELISA), of use forMUC5AC in clinical samples. ELISA experiments are described in theExamples below.

The murine, chimeric, humanized and fully human PAM4 antibodies andfragments thereof described herein are exemplary of anti-pancreaticcancer antibodies of use for diagnostic and/or therapeutic methods. TheExamples below disclose a preferred embodiment of the construction anduse of a humanized PAM4 antibody. Because non-human monoclonalantibodies can be recognized by the human host as a foreign protein, andrepeated injections can lead to harmful hypersensitivity reactions,humanization of a murine antibody sequences can reduce the adverseimmune response that patients may experience. For murine-basedmonoclonal antibodies, this is often referred to as a Human Anti-MouseAntibody (HAMA) response. Preferably some human residues in theframework regions of the humanized anti-pancreatic cancer antibody orfragments thereof are replaced by their murine counterparts. It is alsopreferred that a combination of framework sequences from two differenthuman antibodies is used for V_(H). The constant domains of the antibodymolecule are derived from those of a human antibody.

Antibody Preparation

Monoclonal antibodies for specific antigens may be obtained by methodsknown to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) (hereinafter “Coligan”). Briefly, anti-pancreatic cancer MAbs canbe obtained by injecting mice with a composition comprising a mixture ofpancreatic cancer mucins comprising MUC5AC, or a purified MUC5AC, or apeptide or protein corresponding to an epitope located within the secondto fourth cysteine-rich domains of MUC5AC (amino acid residues1575-2052), more preferably to an epitope located in amino acid residues1575-1725 and 1903-2052 (Cys2 and Cys 4), even more preferably to anepitope located in amino acid residues 1575-1725 (Cys2+), mostpreferably, to an epitope located in Cys2, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to MUC5AC, culturing the clones that produceantibodies to an epitope of MUC5AC as discussed above, and isolatinganti-pancreatic cancer antibodies from the hybridoma cultures.

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques toproduce chimeric or humanized antibodies. Chimerization of murineantibodies and antibody fragments are well known to those skilled in theart. The use of antibody components derived from chimerized monoclonalantibodies reduces potential problems associated with the immunogenicityof murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., ProcNat'l Acad. Sci. USA 86: 3833 (1989), incorporated herein by reference.In general, the V_(K) (variable light chain) and V_(H) (variable heavychain) sequences for murine antibodies can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the V_(H) and V_(K) sequences of the murinePAM4 MAb were cloned by PCR amplification from the hybridoma cells byRT-PCR, and their sequences determined by DNA sequencing. To confirmtheir authenticity, the cloned V_(K) and V_(H) genes can be expressed incell culture as a chimeric Ab as described by Orlandi et al., (ProcNatl. Acad. Sci., USA, 86: 3833, 1989).

In a preferred embodiment, a chimerized PAM4 antibody or antibodyfragment comprises the complementarity-determining regions (CDRs) andframework regions (FR) of a murine PAM4 MAb and the light and heavychain constant regions of a human antibody, wherein the CDRs of thelight chain variable region of the chimerized PAM4 comprises CDR1(SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3(HQWNRYPYT, SEQ ID NO:3); and the CDRs of the heavy chain variableregion of the chimerized PAM4 MAb comprises CDR1 (SYVLH, SEQ ID NO:4);CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ IDNO:6). The use of antibody components derived from chimerized monoclonalantibodies reduces potential problems associated with the immunogenicityof murine constant regions.

Humanization of murine antibodies and antibody fragments is also wellknown to those skilled in the art. Techniques for producing humanizedMAbs are disclosed, for example, by Carter et al., Proc Nat'l Acad. Sci.USA 89: 4285 (1992), Singer et al., J. Immun. 150: 2844 (1992), Mountainet al. Biotechnol Genet Eng Rev. 10:1 (1992), and Coligan at pages10.19.1-10.19.11, each of which is incorporated herein by reference. Forexample, humanized monoclonal antibodies may be produced by transferringmurine complementary determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen substituting selected human residues in the framework regions withtheir the murine FR counterparts. The use of human framework regionsequences, in addition to human constant region sequences, furtherreduces the chance of inducing HAMA reactions.

Humanized antibodies can be designed and constructed as described byLeung et al. (Mol Immunol. 32: 1413 (1995)). Example 3 describes thehumanization process utilized for construction of the hPAM4 MAb.

The nucleotide sequences of the primers used to prepare the hPAM4antibodies are discussed in Example 3, below. In a preferred embodiment,a humanized PAM4 antibody or antibody fragment comprises the light andheavy chain CDR sequences (SEQ ID NO: 1 to SEQ ID NO:6) disclosed above.Also preferred, the FRs of the light and heavy chain variable regions ofthe humanized antibody comprise at least one amino acid substituted fromsaid corresponding FRs of the murine PAM4 MAb.

A fully human antibody, e.g., human PAM4 can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425. For example, a humanantibody can be recovered from a transgenic mouse possessing humanimmunoglobulin loci. The mouse humoral immune system is humanized byinactivating the endogenous immunoglobulin genes and introducing humanimmunoglobulin loci. The human immunoglobulin loci are exceedinglycomplex and comprise a large number of discrete segments which togetheroccupy almost 0.2% of the human genome. To ensure that transgenic miceare capable of producing adequate repertoires of antibodies, largeportions of human heavy- and light-chain loci must be introduced intothe mouse genome. This is accomplished in a stepwise process beginningwith the formation of yeast artificial chromosomes (YACs) containingeither human heavy- or light-chain immunoglobulin loci in germlineconfiguration. Since each insert is approximately 1 Mb in size, YACconstruction requires homologous recombination of overlapping fragmentsof the immunoglobulin loci. The two YACs, one containing the heavy-chainloci and one containing the light-chain loci, are introduced separatelyinto mice via fusion of YAC-containing yeast spheroblasts with mouseembryonic stem cells. Embryonic stem cell clones are then microinjectedinto mouse blastocysts. Resulting chimeric males are screened for theirability to transmit the YAC through their germline and are bred withmice deficient in murine antibody production. Breeding the twotransgenic strains, one containing the human heavy-chain loci and theother containing the human light-chain loci, creates progeny whichproduce human antibodies in response to immunization. However, thesetechniques are not limiting and other methods known in the art forproducing human antibodies, such as the use of phage display, may alsobe utilized to produce human anti-pancreatic cancer antibodies.

Antibodies can be produced by cell culture techniques using methodsknown in the art. In one example transfectoma cultures are adapted toserum-free medium. For production of humanized antibody, cells may begrown as a 500 ml culture in roller bottles using HSFM. Cultures arecentrifuged and the supernatant filtered through a 0.2 m membrane. Thefiltered medium is passed through a protein-A column (1×3 cm) at a flowrate of 1 ml/min. The resin is then washed with about 10 column volumesof PBS and protein A-bound antibody is eluted from the column with 0.1 Mglycine buffer (pH 3.5) containing 10 mM EDTA. Fractions of 1.0 ml arecollected in tubes containing 10 μl of 3 M Tris (pH 8.6), and proteinconcentrations determined from the absorbance at 280/260 nm. Peakfractions are pooled, dialyzed against PBS, and the antibodyconcentrated, for example, with a Centricon 30 filter (Amicon, Beverly,Mass.). The antibody concentration is determined by ELISA and itsconcentration adjusted to about 1 mg/ml using PBS. Sodium azide, 0.01%(w/v), is conveniently added to the sample as preservative.

Antibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A SEPHAROSE®,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Anti-pancreatic cancer MAbs can be characterized by a variety oftechniques that are well-known to those of skill in the art. Forexample, the ability of an antibody to bind to an epitope of MUC5AC asdiscussed above can be verified using an indirect enzyme immunoassay,flow cytometry analysis, ELISA or Western blot analysis.

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a V_(L) domain and a V_(H)domain. The V_(L) and V_(H) domains associate to form a target bindingsite. These two domains are further covalently linked by a peptidelinker (L). A scFv molecule is denoted as either V_(L)-L-V_(H) if theV_(L) domain is the N-terminal part of the scFv molecule, or asV_(H)-L-V_(L) if the V_(H) domain is the N-terminal part of the scFvmolecule. Methods for making scFv molecules and designing suitablepeptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80(1995) and R. E. Bird and B. W. Walker, Single Chain Antibody VariableRegions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional V_(H)-V_(L) pairs.(Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelidheavy chain only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacasmay be immunized with known antigens, such as TNF-α, and VHHs can beisolated that bind to and neutralize the target antigen (Maass et al.,2007). PCR primers that amplify virtually all alpaca VHH codingsequences have been identified and may be used to construct alpaca VHHphage display libraries, which can be used for antibody fragmentisolation by standard biopanning techniques well known in the art (Maasset al., 2007). Commercially available single domain antibodies, alsoknown as nanobodies, may be purchased for example from Ablynx (Ghent,Belgium).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 Kdfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Antibody Fusion Proteins and Multivalent Antibodies

Fusion proteins comprising the anti-pancreatic cancer antibodies ofinterest can be prepared by a variety of conventional procedures,ranging from glutaraldehyde linkage to more specific linkages betweenfunctional groups. The antibodies and/or antibody fragments thatcomprise the fusion proteins described herein are preferably covalentlybound to one another, directly or through a linker moiety, through oneor more functional groups on the antibody or fragment, e.g., amine,carboxyl, phenyl, thiol, or hydroxyl groups. Various conventionallinkers in addition to glutaraldehyde can be used, e.g., diisocyanates,diiosothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides,maleimidehydroxy succinimide esters, and the like.

A simple method for producing fusion proteins is to mix the antibodiesor fragments in the presence of glutaraldehyde. The initial Schiff baselinkages can be stabilized, e.g., by borohydride reduction to secondaryamines. A diiosothiocyanate or carbodiimide can be used in place ofglutaraldehyde as a non-site-specific linker. In one embodiment, anantibody fusion protein comprises an anti-pancreatic cancer MAb, orfragment thereof, wherein the MAb binds to an epitope located within thesecond to fourth cysteine-rich domains of MUC5AC (amino acid residues1575-2052), more preferably to an epitope located in amino acid residues1575-1725 and 1903-2052 (Cys2 and Cys 4), even more preferably to anepitope located in amino acid residues 1575-1725 (Cys2+), mostpreferably, to an epitope located in Cys2. This fusion protein andfragments thereof preferentially bind pancreatic cancer cells. Thismonovalent, monospecific MAb is useful for direct targeting of anantigen, where the MAb is attached to a therapeutic agent, a diagnosticagent, or a combination thereof, and the protein is administereddirectly to a patient.

The fusion proteins may instead comprise at least two anti-pancreaticcancer MAbs that bind to distinct epitopes of MUC5AC. For example, theMAbs can produce antigen specific diabodies, triabodies and tetrabodies,which are multivalent but monospecific to the MUCSAC. The non-covalentassociation of two or more scFv molecules can form functional diabodies,triabodies and tetrabodies. Monospecific diabodies are homodimers of thesame scFv, where each scFv comprises the V_(H) domain from the selectedantibody connected by a short linker to the V_(L) domain of the sameantibody. A diabody is a bivalent dimer formed by the non-covalentassociation of two scFvs, yielding two Fv binding sites. A triabodyresults from the formation of a trivalent trimer of three scFvs,yielding three binding sites, and a tetrabody is a tetravalent tetramerof four scFvs, resulting in four binding sites. Several monospecificdiabodies have been made using an expression vector that contains arecombinant gene construct comprising V_(H)1-linker-V_(L)1. See Holligeret al., Proc Natl. Acad. Sci USA 90: 6444-6448 (1993); Atwell et al.,Molecular Immunology 33: 1301-1302 (1996); Holliger et al., NatureBiotechnology 15: 632-631 (1997); Helfrich et al., Int J Cancer 76:232-239 (1998); Kipriyanov et al., Int J Cancer 77: 763-772 (1998);Holiger et al., Cancer Research 59: 2909-2916 (1999)). Methods ofconstructing scFvs are disclosed in U.S. Pat. No. 4,946,778 (1990) andU.S. Pat. No. 5,132,405 (1992), the Examples section of each of which isincorporated herein by reference. Methods of producing multivalent,monospecific antibodies based on scFv are disclosed in U.S. Pat. No.5,837,242 (1998), U.S. Pat. No. 5,844,094 (1998) and WO-98/44001 (1998),the Examples section of each of which is incorporated herein byreference. The multivalent, monospecific antibody fusion protein bindsto two or more of the same type of epitopes that can be situated on thesame antigen or on separate antigens. The increased valency allows foradditional interaction, increased affinity, and longer residence times.These antibody fusion proteins can be utilized in direct targetingsystems, where the antibody fusion protein is conjugated to atherapeutic agent, a diagnostic agent, or a combination thereof, andadministered directly to a patient in need thereof.

A preferred embodiment is a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites having anaffinity toward a PAM4 target epitope and one or more additional bindingsites for other epitopes associated with pancreatic cancer. This fusionprotein is multispecific because it binds at least two differentepitopes, which can reside on the same or different antigens. Forexample, the fusion protein may comprise more than one antigen bindingsite, the first with an affinity toward an epitope of MUC5AC asdiscussed above and the second with an affinity toward another targetantigen such as TAG-72 or CEA. Another example is a bispecific antibodyfusion protein which may comprise a CA19.9 MAb (or fragment thereof) anda PAM4 MAb (or fragment thereof). Such a fusion protein will have anaffinity toward CA19.9 as well as MUC5AC. The antibody fusion proteinsand fragments thereof can be utilized in direct targeting systems, wherethe antibody fusion protein is conjugated to a therapeutic agent, adiagnostic agent, or a combination thereof, and administered directly toa patient in need thereof.

Another preferred embodiment is a multivalent, multispecific antibodycomprising at least one binding site having affinity toward a PAM4target epitope and at least one hapten binding site having affinitytowards hapten molecules. For example, a bispecific fusion protein maycomprise the 679 MAb (or fragment thereof) and the PAM4 MAb (or fragmentthereof). The monoclonal 679 antibody binds with high affinity tomolecules containing the tri-peptide moiety histamine succinyl glycyl(HSG). Such a bispecific PAM4 antibody fusion protein can be prepared,for example, by obtaining a F(ab′)₂ fragment from 679, as describedabove. The interchain disulfide bridges of the 679 F(ab′)₂ fragment aregently reduced with DTT, taking care to avoid light-heavy chain linkage,to form Fab′-SH fragments. The SH group(s) is (are) activated with anexcess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)b-is-maleimide). The PAM4 MAb isconverted to Fab′-SH and then reacted with the activated 679 Fab′-SHfragment to obtain a bispecific antibody fusion protein. Bispecificantibody fusion proteins such as this one can be utilized in affinityenhancing systems, where the target antigen is pretargeted with thefusion protein and is subsequently targeted with a diagnostic ortherapeutic agent attached to a carrier moiety (targetable construct)containing one or more HSG haptens. In alternative preferredembodiments, a DNL™-based hPAM4-679 construct, such as TF10, may beprepared and used as described in the Examples below.

Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody fusion proteins have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody fusion proteins containing a Fab′ portion specificto each of the original epitopes. General techniques for the preparationof antibody fusion proteins may be found, for example, in Nisonoff etal., Arch Biochem Biophys. 93: 470 (1961), Hammerling et al., J Exp Med.128: 1461 (1968), and U.S. Pat. No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimidehydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g.,an antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen-binding sites. This can be accomplished by, e.g.,linkage to cleaved interchain sulfhydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody that has at lease one free amine function.This results in an initial Schiff base linkage, which is preferablystabilized by reduction to a secondary amine, e.g., by borohydridereduction, to form the final composite. Such site-specific linkages aredisclosed, for small molecules, in U.S. Pat. No. 4,671,958, and forlarger addends in U.S. Pat. No. 4,699,784, the Examples section of eachof which is incorporated herein by reference.

ScFvs with linkers greater than 12 amino acid residues in length (forexample, 15- or 18-residue linkers) allow interactions between the V_(H)and V_(L) domains on the same chain and generally form a mixture ofmonomers, dimers (termed diabodies) and small amounts of higher massmultimers, (Kortt et al., Eur J Biochem. (1994) 221: 151-157). ScFvswith linkers of 5 or less amino acid residues, however, prohibitintramolecular pairing of the V_(H) and V_(L) domains on the same chain,forcing pairing with V_(H) and V_(L) domains on a different chain.Linkers between 3- and 12-residues form predominantly dimers (Atwell etal., Protein Engineering (1999) 12: 597-604). With linkers between 0 and2 residues, trimeric (termed triabodies), tetrameric (termedtetrabodies) or higher oligomeric structures of scFvs are formed;however, the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of the V-domains, in addition tothe linker length.

More recently, a novel technique for construction of mixtures ofantibodies, antibody fragments and/or other effector moieties invirtually any combination has been described in U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787; and 7,666,400, the Examplessection of each of which is incorporated herein by reference. Thetechnique, known generally as DOCK-AND-LOCK™ (DNL™) involves theproduction of fusion proteins that comprise at their N- or C-terminalends one of two complementary peptide sequences, called dimerization anddocking domain (DDD) and anchoring domain (AD) sequences. In preferredembodiments, the DDD sequences are derived from the regulatory subunitsof cAMP-dependent protein kinase and the AD sequence is derived from thesequence of A-kinase anchoring protein (AKAP). The DDD sequences formdimers that bind to the AD sequence, which allows formation of trimers,tetramers, hexamers or any of a variety of other complexes. By attachingeffector moieties, such as antibodies or antibody fragments, to the DDDand AD sequences, complexes may be formed of any selected combination ofantibodies or antibody fragments. The DNL™ complexes may be covalentlystabilized by formation of disulfide bonds or other linkages.

Pretargeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct ortargetable conjugate) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are well known in the art, for example, asdisclosed in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J.Nucl. Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987;Oehr et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. No. 6,077,499; U.S. Pat. No. 6,090,381; U.S. Pat. No.6,472,511; and U.S. Pat. No. 6,962,702.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antigen binding antibody fragment; (2) optionallyadministering to the subject a clearing composition, and allowing thecomposition to clear the antibody from circulation; and (3)administering to the subject the targetable construct, containing one ormore chelated or chemically bound therapeutic or diagnostic agents. Thetechnique may also be utilized for antibody dependent enzyme prodrugtherapy (ADEPT) by administering an enzyme conjugated to a targetableconstruct, followed by a prodrug that is converted into active form bythe enzyme.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art, for example forcombination antibody therapy. Antibodies of use may be commerciallyobtained from a number of known sources. For example, a variety ofantibody secreting hybridoma lines are available from the American TypeCulture Collection (ATCC, Manassas, Va.). A large number of antibodiesagainst various disease targets, including but not limited totumor-associated antigens, have been deposited at the ATCC and/or havepublished variable region sequences and are available for use in theclaimed methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318;7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060;7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498;7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241;6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107;6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645;6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681;6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405;6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511;6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780;6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711;6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307;6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607;6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344;6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294;6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572,856;6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206;6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;5,587,459; 5,443,953; 5,525,338, the Examples section of each of whichis incorporated herein by reference. These are exemplary only and a widevariety of other antibodies and their hybridomas are known in the art.The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20,anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20),lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor),ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1,also known as TROP-2)), KC4 (anti-mucin), MN-14 (anti-carcinoembryonicantigen (anti-CEA, also known as CD66e or CEACAM5), MN-15 or MN-3(anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72(e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membraneantigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (ananti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-MUC5AC) andtrastuzumab (anti-ErbB2). Such antibodies are known in the art (e.g.,U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393;6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403;7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655;7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. Patent ApplicationPubl. No. 20050271671; 20060193865; 20060210475; 20070087001; theExamples section of each incorporated herein by reference.) Specificknown antibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20(U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU-31(U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318), hLL2 (U.S.Pat. No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat.No. 7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.7,541,440), hR1 (U.S. patent application Ser. No. 12/772,645), hRS7(U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440),AB-PG1-XG1-026 (U.S. patent application Ser. No. 11/983,372, depositedas ATCC PTA-4405 and PTA-4406), D2/B (WO 2009/130575), BWA-3(anti-histone H4), LG2-1 (anti-histone H3) and LG2-2 (anti-histone H2B)(U.S. patent application Ser. No. 14/180,646, filed Feb. 14, 2014) thetext of each recited patent or application is incorporated herein byreference with respect to the Figures and Examples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD47, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80,CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4,CXCR4, alpha-fetoprotein (AFP), VEGF (e.g., AVASTIN®, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., ERBITUX®), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733, GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, histone H2B, histone H3, histone H4,insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides,HCG, the HLA-DR antigen to which L243 binds, CD66 antigens, i.e.,CD66a-d or a combination thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B,macrophage migration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4,MUC5AC, placental growth factor (PlGF), PSA (prostate-specific antigen),PSMA, PD-1, PD-L1, TIM-3, LAG-3, matrix metalloproteinase-1 (MMP-1),MMP-2, MMP-7, MMP-9, MMP-14, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1,Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreichantigens, tumor necrosis antigens, tumor angiogenesis antigens, TNF-α,TRAIL receptor (R1 and R2), TROP-2, VEGFR, RANTES, T101, as well ascancer stem cell antigens, complement factors C3, C3a, C3b, C5a, C5, andan oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8). Another useful target for breast cancer therapy is theLIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375:51-9).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Checkpoint inhibitor antibodies have been used primarily in cancertherapy. Immune checkpoints refer to inhibitory pathways in the immunesystem that are responsible for maintaining self-tolerance andmodulating the degree of immune system response to minimize peripheraltissue damage. However, tumor cells can also activate immune systemcheckpoints to decrease the effectiveness of immune response againsttumor tissues. Exemplary checkpoint inhibitor antibodies againstcytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152),programmed cell death protein 1 (PD-1, also known as CD279) andprogrammed cell death 1 ligand 1 (PD-L1, also known as CD274), may beused in combination with one or more other agents to enhance theeffectiveness of immune response against disease cells, tissues orpathogens. Exemplary anti-PD1 antibodies include lambrolizumab (MK-3475,MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK),and pidilizumab (CT-011, CURETECH LTD.). Anti-PD1 antibodies arecommercially available, for example from ABCAM® (AB137132), BIOLEGEND®(EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).Anti-PD-L1 antibodies are also commercially available, for example fromAFFYMETRIX EBIOSCIENCE (MIH1). Exemplary anti-CTLA4 antibodies includeipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Anti-PD1antibodies are commercially available, for example from ABCAM®(AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMOSCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205,MA1-35914). Ipilimumab has recently received FDA approval for treatmentof metastatic melanoma (Wada et al., 2013, J Transl Med 11:89). Morerecently, other checkpoint inhibitory receptors have been identified,including TIM-3 and LAG-3 (Stagg, 2013, Ther Adv Med Oncol 5:169-81).Antibodies against TIM-3 and LAG-3 may also be used in combination withthe anti-MUC5AC antibodies disclosed herein.

Antibodies against matrix metalloproteinases, for example matrixmetalloproteinase-1 (MMP-1), MMP-2, MMP-7, MMP-9 and MMP-14, are also ofuse in combination anti-cancer therapies. (See, e.g., Agarwal A, et al.,Mol Cancer Ther 2008; 7:2746-57; Freije J M, et al. Adv Exp Med Biol2003; 532:91-107; Coticchia C M, et al. Gynecol Oncol 2011; 123:295-300;Boiire D, et al., Cell 2005; 120:303-13; Belotti D, et al., Cancer Res2003; 63:5224-9; Barbolina M V, et al., J Biol Chem 2007; 282:4924-31;Kaimal R, et al., Cancer Res 2013; 73:2457-67; Denzel S, et al, Int JExp Pathol 2012; 93:341-53.)

Other antibodies of use may include anti-histone antibodies and/orantigen-binding fragments thereof, such as the BWA-3 (anti-H4), LG2-1(anti-H3) and LG2-2 (anti-H2B) antibodies. Exemplary anti-histoneantibodies are disclosed, for example, in U.S. patent application Ser.No. 14/180,646, filed Feb. 14, 2014 (the Examples section of which isincorporated herein by reference).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):5556s-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

Antibody Use for Treatment and Diagnosis

Certain embodiments concern methods of diagnosing or treating amalignancy in a subject, comprising administering to the subject ananti-pancreatic cancer MAb, fusion protein or fragment thereof, whereinthe MAb, fusion protein or fragment is bound to at least one diagnosticand/or therapeutic agent. The antibody preferably binds to an epitopelocated within the second to fourth cysteine-rich domains of MUC5AC(amino acid residues 1575-2052), more preferably to an epitope locatedin amino acid residues 1575-1725 and 1903-2052 (Cys2 and Cys 4), evenmore preferably to an epitope located in amino acid residues 1575-1725(Cys2+), most preferably, to an epitope located in Cys2

Also preferred is a method for diagnosing or treating cancer, comprisingadministering to a subject a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites toward anepitope of MUC5AC as discussed above and one or more hapten bindingsites, waiting a sufficient amount of time for non-bound antibody toclear the subject's blood stream; and then administering to the subjecta carrier molecule comprising a diagnostic agent, a therapeutic agent,or a combination thereof, that binds to the hapten-binding site of thelocalized antibody. In a more preferred embodiment, the cancer is anon-endocrine pancreatic cancer.

The use of MAbs for in vitro diagnosis is well-known. See, for example,Carlsson et al., Bio/Technology 7 (6): 567 (1989). For example, MAbs canbe used to detect the presence of a tumor-associated antigen in tissuefrom biopsy samples. MAbs also can be used to measure the amount oftumor-associated antigen in clinical fluid samples, such as blood orserum, using techniques such as radioimmunoassay, enzyme-linkedimmunosorbent assay, and fluorescence immunoassay. In vitro and in vivomethods of diagnosis are discussed in further detail below.

Conjugates of tumor-targeted MAbs and toxins can be used to selectivelykill cancer cells in vivo (Spalding, Bio/Technology 9(8): 701 (1991);Goldenberg, Scientific American Science & Medicine 1(1): 64 (1994)). Forexample, therapeutic studies in experimental animal models havedemonstrated the anti-tumor activity of antibodies carrying cytotoxicradionuclides. (Goldenberg et al., Cancer Res. 41: 4354 (1981), Cheunget al., J. Nat'l Cancer Inst. 77: 739 (1986), and Senekowitsch et al.,J. Nucl. Med. 30: 531 (1989)). In a preferred embodiment, the conjugatecomprises a ⁹⁰Y-labeled hPAM4 antibody. The conjugate may optionally beadministered in conjunction with one or more other therapeutic agents.In a preferred embodiment, ⁹⁰Y-labeled hPAM4 is administered togetherwith gemcitabine or 5-fluorouracil to a patient with pancreatic cancer.In a further preferred embodiment, ⁹⁰Y is conjugated to a DOTA chelatefor attachment to hPAM4. In a more preferred embodiment, the⁹⁰Y-DOTA-hPAM4 is combined with gemcitabine in fractionated dosescomprising a treatment cycle, such as with repeated, lower, less-toxicdoses of gemcitabine combined with lower, fractionated doses of⁹⁰Y-DOTA-hPAM4. Alternatively, a radiolabeled or other conjugated PAM4antibody may be administered in combination with anotherimmunoconjugate, such as an SN-38 conjugated antibody. A particularlypreferred combination is ⁹⁰Y-hPAM4 and SN-38-hRS7 (anti-TROP2 antibody)(see, e.g., U.S. Pat. No. 8,586,050, the Examples section incorporatedherein by reference).

As tolerated, repeated cycles of a fractionated dose schedule areindicated. By way of example, 4 weekly doses of 200 mg/m² of gemcitabineare combined with three weekly doses of 8 mg/m² of ⁹⁰Y-DOTA-hPAM4, withthe latter commencing in the second week of gemcitabine administration,constitutes a single therapy cycle. Still other doses, higher or lowerof each component, may constitute a fractionated dose, which isdetermined by conventional means of assessing hematopoietic toxicity(see, e.g., U.S. Pat. Nos. 6,649,352; 7,112,409; 7,279,289; 7,465,551),since myelosuppressive effects of both agents can be cumulative. Askilled physician in such therapy interventions can adjust these dosesbased on the patient's bone marrow status and general health statusbased on many factors, including prior exposure to myelosuppressivetherapeutic agents. These principles can also apply to combinations ofradiolabeled hPAM4 with other therapeutic agents, includingradiosensitizing drugs such as 5-fluorouracil and cisplatin.

Chimeric, humanized and human antibodies and fragments thereof have beenused for in vivo therapeutic and diagnostic methods. Accordinglycontemplated is a method of delivering a diagnostic or therapeuticagent, or a combination thereof, to a target comprising (i) providing acomposition that comprises an anti-pancreatic cancer antibody orfragment thereof, such as a chimeric, humanized or human PAM4 antibody,conjugated to at least one diagnostic and/or therapeutic agent and (ii)administering to a subject the diagnostic or therapeutic antibodyconjugate. In a preferred embodiment, the anti-pancreatic cancerantibodies and fragments thereof are humanized or fully human.

Another embodiment concerns a method for treating a malignancycomprising administering a naked or conjugated anti-pancreatic cancerantibody, antibody fragment or fusion protein that binds to an epitopelocated within the second to fourth cysteine-rich domains of MUC5AC(amino acid residues 1575-2052), more preferably to an epitope locatedin amino acid residues 1575-1725 and 1903-2052 (Cys2 and Cys 4), evenmore preferably to an epitope located in amino acid residues 1575-1725(Cys2+), most preferably, to an epitope located in Cys2, such as a PAM4antibody, either alone or in conjunction with one or more othertherapeutic agents. The other therapeutic agent may be added before,simultaneously with or after the antibody. In a preferred embodiment,the therapeutic agent is gemcitabine, and in a more preferredembodiment, gemcitabine is given with the hPAM4 radioconjugate in afractionated dose schedule at lower doses than the conventional800-1,000 mg/m² doses of gemcitabine given weekly for 6 weeks. Forexample, when combined with fractionated therapeutic doses of ⁹⁰Y-PAM4,repeated fractionated doses intended to function as a radiosensitizingagent of 200-380 mg/m² gemcitabine are infused. The skilled artisan willrealize that the antibodies, fusion proteins and/or fragments thereofdescribed and claimed herein may be administered with any known ordescribed therapeutic agent, including but not limited to heat shockprotein 90 (Hsp90).

In another form of multimodal therapy, subjects receive immunoconjugatesin conjunction with standard cancer chemotherapy. For example, “CVB”(1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and 150-200 mg/m²carmustine) is a regimen used to treat non-Hodgkin's lymphoma. Patti etal., Eur. J. Haematol. 51: 18 (1993). Other suitable combinationchemotherapeutic regimens are well-known to those of skill in the art.See, for example, Freedman et al., “Non-Hodgkin's Lymphomas,” in CANCERMEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.), pages 2028-2068(Lea & Febiger 1993). As an illustration, first generationchemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

The present invention contemplates the administration of anti-pancreaticcancer antibodies and fragments thereof, including fusion proteins andfragments thereof, alone, as a naked antibody or antibody fragment, oradministered as a multimodal therapy. Preferably, the antibody is ahumanized or fully human PAM4 antibody or fragment thereof. Multimodaltherapies further include immunotherapy with a naked anti-pancreaticcancer antibody supplemented with administration of other antibodies inthe form of naked antibodies, fusion proteins, or as immunoconjugates.For example, a humanized or fully human PAM4 antibody may be combinedwith another naked antibody, or a humanized PAM4 or other antibodyconjugated to an isotope, one or more chemotherapeutic agents,cytokines, toxins or a combination thereof. For example, the presentinvention contemplates treatment of a naked or conjugated PAM4 antibodyor fragments thereof before, in combination with, or after otherpancreatic tumor associated antibodies such as CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, anti-Le^(a) antibodies, and antibodies to other Lewisantigens (e.g., Le(y)), as well as antibodies against carcinoembryonicantigen (CEA or CEACAM5), CEACAM6, colon-specific antigen-p (CSAp),MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC16, MUC17, HLA-DR, CD40, CD74, CD138,HER2/neu, EGFR, EGP-1, EGP-2, angiogenesis factors (e.g., VEGF, PlGF),insulin-like growth factor (IGF), tenascin, platelet-derived growthfactor, and IL-6, as well as products of oncogenes (e.g., bcl-2, Kras,p53), cMET, and antibodies against tumor necrosis substances.

These solid tumor antibodies may be naked or conjugated to, inter alia,drugs, toxins, isotopes, radionuclides or immunomodulators. Manydifferent antibody combinations may be constructed and used in eithernaked or conjugated form. Alternatively, different naked antibodycombinations may be employed for administration in combination withother therapeutic agents, such as a cytotoxic drug or with radiation,given consecutively, simultaneously, or sequentially.

Administration of the antibodies and their fragments can be effected byintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, perfusion through a regionalcatheter, or direct intralesional injection. When administering theantibody by injection, the administration may be by continuous infusionor by single or multiple boluses.

The immunoconjugate of the present invention can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the antibody of the present inventionis infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours. For example, thefirst 25-50 mg could be infused within 30 minutes, preferably even 15min, and the remainder infused over the next 2-3 hrs. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate or antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

More generally, the dosage of an administered immunoconjugate for humanswill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history. Itmay be desirable to provide the recipient with a dosage ofimmunoconjugate, antibody fusion protein that is in the range of fromabout 1 mg/kg to 25 mg/kg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. A dosage of 1-20 mg/kg for a 70 kg patient, for example, is70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosage may berepeated as needed, for example, once per week for 4-10 weeks, once perweek for 8 weeks, or once per week for 4 weeks. It may also be givenless frequently, such as every other week for several months, or monthlyor quarterly for many months, as needed in a maintenance therapy.

Alternatively, an antibody may be administered as one dosage every 2 or3 weeks, repeated for a total of at least 3 dosages. Or, the antibodiesmay be administered twice per week for 4-6 weeks. If the dosage islowered to approximately 200-300 mg/m² (340 mg per dosage for a 1.7-mpatient, or 4.9 mg/kg for a 70 kg patient), it may be administered onceor even twice weekly for 4 to 10 weeks. Alternatively, the dosageschedule may be decreased, namely every 2 or 3 weeks for 2-3 months. Ithas been determined, however, that even higher doses, such as 20 mg/kgonce weekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

Immunoconjugates

Anti-pancreatic cancer antibodies and fragments thereof may beconjugated to at least one therapeutic and/or diagnostic agent fortherapy or diagnosis. For immunotherapy, the objective is to delivercytotoxic doses of radioactivity, toxin, antibody and/or drug to targetcells, while minimizing exposure to non-target tissues. Preferably,anti-pancreatic cancer antibodies are used to diagnose and/or treatpancreatic tumors.

Any of the antibodies, antibody fragments and fusion proteins can beconjugated with one or more therapeutic or diagnostic agents, using avariety of techniques known in the art. One or more therapeutic ordiagnostic agents may be attached to each antibody, antibody fragment orfusion protein, for example by conjugating an agent to a carbohydratemoiety in the Fc region of the antibody. If the Fc region is absent (forexample with certain antibody fragments), it is possible to introduce acarbohydrate moiety into the light chain variable region of either anantibody or antibody fragment to which a therapeutic or diagnostic agentmay be attached. See, for example, Leung et al., J Immunol. 154: 5919(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al.,U.S. Pat. No. 6,254,868, the Examples section of each patentincorporated herein by reference.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int J Cancer 41: 832 (1988); Shih et al., IntJ Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313, theExamples section of which is incorporated herein by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality oftherapeutic agents, such as peptides or drugs. This reaction results inan initial Schiff base (imine) linkage, which can be stabilized byreduction to a secondary amine to form the final conjugate.

Antibody fusion proteins or multispecific antibodies comprise two ormore antibodies or fragments thereof, each of which may be attached toat least one therapeutic agent and/or diagnostic agent. Accordingly, oneor more of the antibodies or fragments thereof of the antibody fusionprotein can have more than one therapeutic and/or diagnostic agentattached. Further, the therapeutic agents do not need to be the same butcan be different therapeutic agents, for example, one can attach a drugand a radioisotope to the same fusion protein. For example, an IgG canbe radiolabeled with ¹³¹I and attached to a drug. The ¹³¹I can beincorporated into the tyrosine of the IgG and the drug attached to theepsilon amino group of the IgG lysines. Both therapeutic and diagnosticagents also can be attached to reduced SH groups and to the carbohydrateside chains of antibodies. Alternatively, a bispecific antibody maycomprise one antibody or fragment thereof against a disease antigen andanother against a hapten attached to a targetable construct, for use inpretargeting techniques as discussed above.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such agents can be attached to the antibody component usinga heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995).

Click Chemistry

An alternative method for attaching chelating moieties, drugs or otherfunctional groups to an antibody, fragment or fusion protein involvesuse of click chemistry reactions. The click chemistry approach wasoriginally conceived as a method to rapidly generate complex substancesby joining small subunits together in a modular fashion. (See, e.g.,Kolb et al., 2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust JChem 60:384-95.) Various forms of click chemistry reaction are known inthe art, such as the Huisgen 1,3-dipolar cycloaddition copper catalyzedreaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which isoften referred to as the “click reaction.” Other alternatives includecycloaddition reactions such as the Diels-Alder, nucleophilicsubstitution reactions (especially to small strained rings like epoxyand aziridine compounds), carbonyl chemistry formation of urea compoundsand reactions involving carbon-carbon double bonds, such as alkynes inthiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is a 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach chelating moieties toantibodies or other targeting molecules in vitro.

Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially, or advantageously conjugated to the antibodies of theinvention, for example, drugs, toxins, oligonucleotides (e.g., siRNA),immunomodulators, hormones, hormone antagonists, enzymes, enzymeinhibitors, radionuclides, angiogenesis inhibitors, pro-apoptoticagents, etc. The therapeutic agents recited here are those agents thatare useful for either conjugated to an antibody, fragment or fusionprotein or for administration separately with a naked antibody asdescribed above.

Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins,taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2inhibitors, antimitotics, antiangiogenic and apoptotic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinaseinhibitors, and others from these and other classes of anticanceragents.

Other useful cancer chemotherapeutic drugs include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins andhormones. Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

Specific drugs of use may include 5-fluorouracil, afatinib, aplidin,azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291,bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan,calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors,irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans,crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib,dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2-PDOX), pro-2PDOX, cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to hPAM4 or otheranti-pancreatic cancer antibodies, for example as disclosed in U.S. Pat.No. 7,591,994; and U.S. patent application Ser. No. 11/388,032, filedMar. 23, 2006, the Examples section of each of which is incorporatedherein by reference.

In another preferred embodiment, prodrug forms of 2-PDOX, as disclosedin U.S. patent application Ser. No. 14/175,089 (the Examples section ofwhich is incorporated herein by reference) may be used as animmunoconjugate with an anti-pancreatic cancer antibody that binds to anepitope of MUC5AC as discussed above.

In another preferred embodiment, an hPAM4 antibody is given withgemcitabine, which may be given before, after, or concurrently with anaked or conjugated chimeric, humanized or human PAM4 antibody.Preferably, the conjugated hPAM4 antibody or antibody fragment isconjugated to a radionuclide.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate of the anti-pancreatic cancer andhPAM4 antibodies. Other toxins suitably employed in the preparation ofsuch conjugates or other fusion proteins, include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, ranpirnase, Pseudomonasexotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al.,Cell 47:641 (1986), Goldenberg, C A—A Cancer Journal for Clinicians44:43 (1994), Sharkey and Goldenberg, C A—A Cancer Journal forClinicians 56:226 (2006). Additional toxins suitable for use are knownto those of skill in the art and are disclosed in U.S. Pat. No.6,077,499, the Examples section of which is incorporated herein byreference.

An immunomodulator, such as a cytokine, may also be conjugated to, orform the therapeutic agent portion of the immunoconjugate, or may beadministered with, but unconjugated to, an antibody, antibody fragmentor fusion protein. The fusion protein may comprise one or moreantibodies or fragments thereof binding to different antigens. Forexample, the fusion protein may bind to an epitope of MUC5AC asdiscussed above as well as to immunomodulating cells or factors.Alternatively, subjects can receive a naked antibody, antibody fragmentor fusion protein and a separately administered cytokine, which can beadministered before, concurrently or after administration of the nakedantibodies. As used herein, the term “immunomodulator” includes acytokine, a lymphokine, a monokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepaticgrowth factor, prostaglandin, fibroblast growth factor, prolactin,placental lactogen, OB protein, a transforming growth factor (TGF),TGF-α, TGF-β, insulin-like growth factor (IGF), erythropoietin,thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, amullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,interleukin (IL), granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interfλeron-γ, S1 factor, IL-1, IL-1cc, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-21 and IL-25, LIF, kit-ligand, FLT-3,angiostatin, thrombospondin, endostatin and lymphotoxin.

The therapeutic agent may comprise one or more radioactive isotopesuseful for treating diseased tissue. Particularly useful therapeuticradionuclides include, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi,²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb and ²²⁷Th. The therapeuticradionuclide preferably has a decay energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5-hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to anantibody using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA).Alternatively, ⁹⁰Y, which emits an energetic beta particle, can becoupled to an antibody, antibody fragment or fusion protein, usingdiethylenetriaminepentaacetic acid (DTPA), or more preferably usingDOTA. Methods of conjugating ⁹⁰Y to antibodies or targetable constructsare known in the art and any such known methods may be used. (See, e.g.,U.S. Pat. No. 7,259,249, the Examples section of which is incorporatedherein by reference. See also Linden et al., Clin Cancer Res.11:5215-22, 2005; Sharkey et al., J Nucl Med. 46:620-33, 2005; Sharkeyet al., J Nucl Med. 44:2000-18, 2003.)

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated antibody or antibody fragment. For example, theradiosensitizer can be used in combination with a radiolabeled antibodyor antibody fragment. The addition of the radiosensitizer can result inenhanced efficacy when compared to treatment with the radiolabeledantibody or antibody fragment alone. Radiosensitizers are described inD. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRCPress (1995). Other typical radionsensitizers of interest for use withthis technology include gemcitabine, 5-fluorouracil, and cisplatin, andhave been used in combination with external irradiation in the therapyof diverse cancers, including pancreatic cancer. Therefore, we havestudied the combination of gemcitabine at what is believed to beradiosensitizing doses (once weekly 200 mg/m² over 4 weeks) ofgemcitabine combined with fractionated doses of ⁹⁰Y-hPAM4, and haveobserved objective evidence of pancreatic cancer reduction after asingle cycle of this combination that proved to be well-tolerated (nograde 3-4 toxicities by NCI-CTC v. 3 standard).

Antibodies or fragments thereof that have a boron addend-loaded carrierfor thermal neutron activation therapy will normally be affected insimilar ways. However, it will be advantageous to wait untilnon-targeted immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an anti-idiotypic antibodythat binds to the anti-pancreatic cancer antibody. See U.S. Pat. No.4,624,846 for a description of this general principle. For example,boron addends such as carboranes, can be attached to antibodies.Carboranes can be prepared with carboxyl functions on pendant sidechains, as is well-known in the art. Attachment of carboranes to acarrier, such as aminodextran, can be achieved by activation of thecarboxyl groups of the carboranes and condensation with amines on thecarrier. The intermediate conjugate is then conjugated to the antibody.After administration of the antibody conjugate, a boron addend isactivated by thermal neutron irradiation and converted to radioactiveatoms which decay by alpha-emission to produce highly toxic, short-rangeeffects.

Interference RNA

Another type of therapeutic agent is RNAi or siRNA. RNA interference(RNAi) is mediated by the RNA-induced silencing complex (RISC) and isinitiated by short double-stranded RNA molecules that interact with thecatalytic RISC component argonaute (Rand et al., 2005, Cell 123:621-29).Types of RNAi molecules include microRNA (miRNA) and small interferingRNA (siRNA). RNAi species can bind with messenger RNA (mRNA) throughcomplementary base-pairing and inhibits gene expression bypost-transcriptional gene silencing. Upon binding to a complementarymRNA species, RNAi induces cleavage of the mRNA molecule by theargonaute component of RISC. Among other characteristics, miRNA andsiRNA differ in the degree of specificity for particular gene targets,with siRNA being relatively specific for a particular target gene andmiRNA inhibiting translation of multiple mRNA species.

Therapeutic use of RNAi by inhibition of selected gene expression hasbeen attempted for a variety of disease states, such as maculardegeneration and respiratory syncytial virus infection (Sah, 2006, LifeSci 79:1773-80). It has been suggested that siRNA functions in host celldefenses against viral infection and siRNA has been widely examined asan approach to antiviral therapy (see, e.g., Zhang et al., 2004, NatureMed 11:56-62; Novina et al., 2002, Nature Med 8:681-86; Palliser et al.,2006, Nature 439:89-94). The use of siRNA for cancer therapy has alsobeen attempted. Fujii et al. (2006, Int J Oncol 29:541-48) transfectedHPV positive cervical cancer cells with siRNA against HPV E6 and E7 andsuppressed tumor growth. siRNA-mediated knockdown of metadherinexpression in breast cancer cells was reported to inhibit experimentallung metastasis (Brown and Ruoslahti, 2004, Cancer Cell 5:365-74).

Attempts have been made to provide targeted delivery of siRNA to reducethe potential for off-target toxicity. Song et al. (2005, Nat Biotechnol23:709-17) used protamine-conjugated Fab fragments against HIV envelopeprotein to deliver siRNA to circulating cells. Schiffelers et al. (2004,Nucl Acids Res 32:e149) conjugated RGD peptides to nanoparticles todeliver anti-VEGFR2 siRNA to tumors and inhibited tumor angiogenesis andgrowth rate in nude mice. Dickerson et al. (2010, Cancer 10:10) usednanogels functionalized with anti-EphA2 receptor peptides tochemosensitize ovarian cancer cells with siRNA against EGFR.Dendrimer-conjugated magnetic nanoparticles have been applied to thetargeted delivery of antisense survivin oligodeoxynucleotides (Pan etal., 2007, Cancer Res 67:8156-63).

The skilled artisan will realize that any siRNA or interference RNAspecies may be attached to the subject antibodies. siRNA and RNAispecies against a wide variety of targets are known in the art, and anysuch known oligonucleotide species may be utilized in the claimedmethods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), andapolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of eachof which is incorporated herein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Mirus Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either an siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL™ complexes.

Exemplary siRNA species that have been reported are listed in Table 1.Although siRNA is delivered as a double-stranded molecule, forsimplicity only the sense strand sequences are shown in Table 1.

TABLE 1 Exemplary siRNA Sequences Target Sequence SEQ ID NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 22 VEGF R2 AAGCTCAGCACACAGAAAGAC SEQ IDNO: 23 CXCR4 UAAAAUCUUCCUGCCCACCdTdT SEQ ID NO: 24 CXCR4GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 25 PPARC1 AAGACCAGCCUCUUUGCCCAG SEQID NO: 26 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 27 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 28 E1A binding proteinUGACACAGGCAGGCUUGACUU SEQ ID NO: 29 Plasminogen GGTGAAGAAGGGCGTCCAA SEQID NO: 30 activator K-ras GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID NO: 31CAAGAGACTCGCCAACAGCTCCAACT TTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAG SEQID NO: 32 Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 33Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 34 Bcl-XUAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO: 35 Raf-1 TTTGAATATCTGTGCTGAGAACACASEQ ID NO: 36 GTTCTCAGCACAGATATTCTTTTT Heat shockaatgagaaaagcaaaaggtgccctgtctc SEQ ID NO: 37 transcription factor 2IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 38 ThioredoxinAUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 39 CD44 GAACGAAUCCUGAAGACAUCU SEQ IDNO: 40 MMP14 AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO: 41 MAPKAPK2UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 42 FGFR1 AAGTCGGACGCAACAGAGAAA SEQ IDNO: 43 ERBB2 CUACCUUUCUACGGACGUGdTdT SEQ ID NO: 44 BCL2L1CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 45 ABL1 TTAUUCCUUCUUCGGGAAGUC SEQ IDNO: 46 CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO: 47 CD9GAGCATCTTCGAGCAAGAA SEQ ID NO: 48 CD151 CATGTGGCACCGTTTGCCT SEQ ID NO:49 Caspase 8 AACTACCAGAAAGGTATACCT SEQ ID NO: 50 BRCA1UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 51 p53 GCAUGAACCGGAGGCCCAUTT SEQ IDNO: 52 CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO: 53

Diagnostic Agents

In the context of this application, the terms “diagnosis” or “detection”can be used interchangeably. Whereas diagnosis usually refers todefining a tissue's specific histological status, detection recognizesand locates a tissue, lesion or organism containing a particularantigen.

The subject antibodies and fragments can be detectably labeled bylinking the antibody to an enzyme. When the antibody-enzyme conjugate isincubated in the presence of the appropriate substrate, the enzymemoiety reacts with the substrate to produce a chemical moiety which canbe detected, for example, by spectrophotometric, fluorometric or visualmeans. Examples of enzymes that can be used to detectably label antibodyinclude malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase,alpha-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase and acetylcholinesterase.

The immunoconjugate may comprise one or more radioactive isotopes usefulfor detecting diseased tissue. Particularly useful diagnosticradionuclides include, but are not limited to, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F,⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O,¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, orother gamma-, beta-, or positron-emitters, preferably with a decayenergy in the range of 20 to 4,000 keV, more preferably in the range of25 to 4,000 keV, and even more preferably in the range of 25 to 1,000keV, and still more preferably in the range of 70 to 700 keV. Totaldecay energies of useful positron-emitting radionuclides are preferably<2,000 keV, more preferably under 1,000 keV, and most preferably <700keV. Radionuclides useful as diagnostic agents utilizing gamma-raydetection include, but are not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu,⁶⁷Ga, ⁷⁵Se, ⁹⁷Ru, ^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb,¹⁹⁷Hg, and ²⁰¹Tl. Decay energies of useful gamma-ray emittingradionuclides are preferably 20-2000 keV, more preferably 60-600 keV,and most preferably 100-300 keV.

Methods of diagnosing cancer in a subject may be accomplished byadministering a diagnostic immunoconjugate and detecting the diagnosticlabel attached to an immunoconjugate that is localized to a cancer ortumor. The antibodies, antibody fragments and fusion proteins may beconjugated to the diagnostic agent or may be administered in apretargeting technique using targetable constructs attached to adiagnostic agent. Radioactive agents that can be used as diagnosticagents are discussed above. A suitable non-radioactive diagnostic agentis a contrast agent suitable for magnetic resonance imaging, X-rays,computed tomography or ultrasound. Magnetic imaging agents include, forexample, non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs. See U.S. Ser. No. 09/921,290 (nowabandoned) filed on Oct. 10, 2001, the Examples section of which isincorporated herein by reference. Other imaging agents such as PETscanning nucleotides, preferably ¹⁸F, may also be used.

Contrast agents, such as MRI contrast agents, including, for example,gadolinium ions, lanthanum ions, dysprosium ions, iron ions, manganeseions or other comparable labels, CT contrast agents, and ultrasoundcontrast agents may be used as diagnostic agents. Paramagnetic ionssuitable for use include chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III), withgadolinium being particularly preferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II) and bismuth (III).Fluorescent labels include rhodamine, fluorescein and renographin.Rhodamine and fluorescein are often linked via an isothiocyanateintermediate.

Metals are also useful in diagnostic agents, including those formagnetic resonance imaging techniques. These metals include, but are notlimited to: gadolinium, manganese, iron, chromium, copper, cobalt,nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to load an antibody with radioactive metals or paramagneticions, it may be necessary to react it with a reagent having a long tailto which are attached a multiplicity of chelating groups for binding theions. Such a tail can be a polymer such as a polylysine, polysaccharide,or other derivatized or derivatizable chain having pendant groups towhich can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates are coupled to an antibody, fusion protein, or fragmentsthereof using standard chemistries. The chelate is normally linked tothe antibody by a group which enables formation of a bond to themolecule with minimal loss of immunoreactivity and minimal aggregationand/or internal cross-linking. Other, more unusual, methods and reagentsfor conjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates”, issued Apr. 25,1989, the Examples section of which is incorporated herein by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 20 to 2,000 keV. The same chelates, whencomplexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA,and TETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates such as macrocyclic polyethers, which are of interest forstably binding nuclides, such as 223Ra for RAIT are encompassed by theinvention.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

The antibodies, antibody fragments and fusion proteins also can belabeled with a fluorescent compound. The presence of afluorescent-labeled MAb is determined by exposing the antibody to lightof the proper wavelength and detecting the resultant fluorescence.Fluorescent labeling compounds include Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, fluorescein isothiocyanate, fluorescamine, HEX,6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, OregonGreen 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalicacid, isophthalic acid, cresyl fast violet, cresyl blue violet,brilliant cresyl blue, para-aminobenzoic acid, erythrosine,phthalocyanines, phthaldehyde, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red. Fluorescently-labeled antibodiesare particularly useful for flow cytometry analysis, but can also beused in endoscopic and intravascular detection methods.

Alternatively, the antibodies, antibody fragments and fusion proteinscan be detectably labeled by coupling the antibody to a chemiluminescentcompound. The presence of the chemiluminescent-tagged MAb is determinedby detecting the presence of luminescence that arises during the courseof a chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label the antibodiesand fragments there. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Bioluminescent compounds that are useful for labelinginclude luciferin, luciferase and aequorin.

Accordingly, a method of diagnosing a malignancy in a subject isdescribed, comprising performing an in vitro diagnosis assay on aspecimen (fluid, tissue or cells) from the subject with a compositioncomprising an anti-pancreatic cancer MAb, fusion protein or fragmentthereof. Immunohistochemistry can be used to detect the presence of PAM4antigen in a cell or tissue by the presence of bound antibody.Preferably, the malignancy that is being diagnosed is a cancer. Mostpreferably, the cancer is pancreatic cancer.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated to a subject antibody. For example, a therapeutically usefulimmunoconjugate can be obtained by conjugating a photoactive agent ordye to an antibody fusion protein. Fluorescent compositions, such asfluorochrome, and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion. In therapy, thishas been termed photoradiation, phototherapy, or photodynamic therapy(Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES(Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)).Moreover, monoclonal antibodies have been coupled with photoactivateddyes for achieving phototherapy. Mew et al., J. Immunol. 130:1473(1983); idem., Cancer Res. 45:4380 (1985); Oseroff et al., Proc Natl.Acad. Sci. USA 83:8744 (1986); idem., Photochem. Photobiol. 46:83(1987); Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta etal., Lasers Surg. Med. 9:422 (1989); Pelegrin et al., Cancer 67:2529(1991).

Fluorescent and radioactive agents conjugated to antibodies or used inbispecific, pretargeting methods, are particularly useful forendoscopic, intraoperative or intravascular detection of the targetedantigens associated with diseased tissues or clusters of cells, such asmalignant tumors, as disclosed in Goldenberg U.S. Pat. Nos. 5,716,595;6,096,289 and 6,387,350, the Examples section of each incorporatedherein by reference, particularly with gamma-, beta- andpositron-emitters. Endoscopic applications may be used when there isspread to a structure that allows an endoscope, such as the colon.Radionuclides useful for positron emission tomography include, but arenot limited to: F-18, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68,As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120,and I-124. Total decay energies of useful positron-emittingradionuclides are preferably <2,000 keV, more preferably under 1,000keV, and most preferably <700 keV. Radionuclides useful as diagnosticagents utilizing gamma-ray detection include, but are not limited to:Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-111,In-114m, I-123, I-125, I-131, Yb-169, Hg-197, and Tl-201. Decay energiesof useful gamma-ray emitting radionuclides are preferably 20-2000 keV,more preferably 60-600 keV, and most preferably 100-300 keV.

In Vitro Diagnosis

The present invention contemplates the use of anti-pancreatic cancerantibodies to screen biological samples in vitro for the presence of thePAM4 antigen. In such immunoassays, the antibody, antibody fragment orfusion protein may be utilized in liquid phase or bound to a solid-phasecarrier, as described below. For purposes of in vitro diagnosis, anytype of antibody such as murine, chimeric, humanized or human may beutilized, since there is no host immune response to consider.

One example of a screening method for determining whether a biologicalsample contains MUC5AC is the radioimmunoassay (RIA). For example, inone form of RIA, the substance under test is mixed with PAM4 MAb in thepresence of radiolabeled MUC5AC. In this method, the concentration ofthe test substance will be inversely proportional to the amount oflabeled MUC5AC bound to the MAb and directly related to the amount offree, labeled MUC5AC. Other suitable screening methods will be readilyapparent to those of skill in the art.

Alternatively, in vitro assays can be performed in which ananti-pancreatic cancer antibody, antibody fragment or fusion protein isbound to a solid-phase carrier. For example, MAbs can be attached to apolymer, such as aminodextran, in order to link the MAb to an insolublesupport such as a polymer-coated bead, a plate or a tube.

Other suitable in vitro assays will be readily apparent to those ofskill in the art. The specific concentrations of detectably labeledantibody and MUC5AC, the temperature and time of incubation, as well asother assay conditions may be varied, depending on various factorsincluding the concentration of MUC5AC in the sample, the nature of thesample, and the like. The binding activity of a sample ofanti-pancreatic cancer antibody may be determined according towell-known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

The presence of the PAM4 antigen in a biological sample can bedetermined using an enzyme-linked immunosorbent assay (ELISA) (e.g.,Gold et al. J Clin Oncol. 24:252-58, 2006). In the direct competitiveELISA, a pure or semipure antigen preparation is bound to a solidsupport that is insoluble in the fluid or cellular extract being testedand a quantity of detectably labeled soluble antibody is added to permitdetection and/or quantitation of the binary complex formed betweensolid-phase antigen and labeled antibody.

In contrast, a “double-determinant” ELISA, also known as a “two-siteELISA” or “sandwich assay,” requires small amounts of antigen and theassay does not require extensive purification of the antigen. Thus, thedouble-determinant ELISA is preferred to the direct competitive ELISAfor the detection of an antigen in a clinical sample. See, for example,the use of the double-determinant ELISA for quantitation of the c-myconcoprotein in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);Spandidos et al., AntiCancer Res. 9: 821 (1989).

In a double-determinant ELISA, a quantity of unlabeled MAb or antibodyfragment (the “capture antibody”) is bound to a solid support, the testsample is brought into contact with the capture antibody, and a quantityof detectably labeled soluble antibody (or antibody fragment) is addedto permit detection and/or quantitation of the ternary complex formedbetween the capture antibody, antigen, and labeled antibody. In thepresent context, an antibody fragment is a portion of an anti-pancreaticcancer MAb that binds to an epitope of MUC5AC. Methods of performing adouble-determinant ELISA are well-known. See, for example, Field et al.,supra, Spandidos et al., supra, and Moore et al., “Twin-Site ELISAs forfos and myc Oncoproteins Using the AMPAK System,” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 273-281 (The Humana Press, Inc. 1992).

In the double-determinant ELISA, the soluble antibody or antibodyfragment must bind to a MUC5AC epitope that is distinct from the epitoperecognized by the capture antibody. The double-determinant ELISA can beperformed to ascertain whether the PAM4 antigen is present in a biopsysample. Alternatively, the assay can be performed to quantitate theamount of MUC5AC that is present in a clinical sample of body fluid. Thequantitative assay can be performed by including dilutions of purifiedMUC5AC.

The anti-pancreatic cancer MAbs, fusion proteins, and fragments thereofalso are suited for the preparation of an assay kit. Such a kit maycomprise a carrier means that is compartmentalized to receive in closeconfinement one or more container means such as vials, tubes and thelike, each of said container means comprising the separate elements ofthe immunoassay.

The subject antibodies, antibody fragments and fusion proteins also canbe used to detect the presence of the PAM4 antigen in tissue sectionsprepared from a histological specimen. Such in situ detection can beused to determine the presence of MUC5AC and to determine thedistribution of MUC5AC in the examined tissue. In situ detection can beaccomplished by applying a detectably-labeled antibody to frozen tissuesections. Studies indicate that the PAM4 antigen is preserved inparaffin-embedded sections. General techniques of in situ detection arewell-known to those of ordinary skill. See, for example, Ponder, “CellMarking Techniques and Their Application,” in MAMMALIAN DEVELOPMENT: APRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987), and Coligan atpages 5.8.1-5.8.8.

Antibodies, antibody fragments and fusion proteins can be detectablylabeled with any appropriate marker moiety, for example, a radioisotope,an enzyme, a fluorescent label, a dye, a chromogen, a chemiluminescentlabel, a bioluminescent labels or a paramagnetic label.

The marker moiety can be a radioisotope that is detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety in thepresent description refers to a molecule that will generate a signalunder predetermined conditions. Examples of marker moieties includeradioisotopes, enzymes, fluorescent labels, chemiluminescent labels,bioluminescent labels and paramagnetic labels.

The binding of marker moieties to anti-pancreatic cancer antibodies canbe accomplished using standard techniques known to the art. Typicalmethodology in this regard is described by Kennedy et al., Clin ChimActa 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1 (1977), Shih etal., Int J Cancer 46: 1101 (1990).

The above-described in vitro and in situ detection methods may be usedto assist in the diagnosis or staging of a pathological condition. Forexample, such methods can be used to detect tumors that express the PAM4antigen such as pancreatic cancer.

In Vivo Diagnosis/Detection

Various methods of in vivo diagnostic imaging with radiolabeled MAbs arewell-known. In the technique of immunoscintigraphy, for example,antibodies are labeled with a gamma-emitting radioisotope and introducedinto a patient. A gamma camera is used to detect the location anddistribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).

For diagnostic imaging, radioisotopes may be bound to antibody eitherdirectly or indirectly by using an intermediary functional group. Usefulintermediary functional groups include chelators such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.For example, see Shih et al., supra, and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound toanti-pancreatic cancer antibody and are appropriate for diagnosticimaging include ^(99m)Tc, ¹¹¹In and ¹⁸F.

The subject antibodies, antibody fragments and fusion proteins also canbe labeled with paramagnetic ions and a variety of radiological contrastagents for purposes of in vivo diagnosis. Contrast agents that areparticularly useful for magnetic resonance imaging comprise gadolinium,manganese, dysprosium, lanthanum, or iron ions. Additional agentsinclude chromium, copper, cobalt, nickel, rhenium, europium, terbium,holmium, or neodymium. Antibodies and fragments thereof can also beconjugated to ultrasound contrast/enhancing agents. For example, oneultrasound contrast agent is a liposome. Also preferred, the ultrasoundcontrast agent is a liposome that is gas filled.

In a preferred embodiment, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In anotherpreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome.

Pharmaceutically Suitable Excipients

Additional pharmaceutical methods may be employed to control theduration of action of an anti-pancreatic cancer antibody in atherapeutic application. Control release preparations can be preparedthrough the use of polymers to complex or adsorb the antibody, antibodyfragment or fusion protein. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan antibody, antibody fragment or fusion protein from such a matrixdepends upon the molecular weight of the antibody, antibody fragment orfusion protein, the amount of antibody within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The antibodies, fragments thereof or fusion proteins to be delivered toa subject can comprise one or more pharmaceutically suitable excipients,one or more additional ingredients, or some combination of these. Theantibody can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate ornaked antibody is combined in a mixture with a pharmaceutically suitableexcipient. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate or naked antibody can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The immunoconjugate, naked antibody, fragment thereof or fusion proteinmay also be administered to a mammal subcutaneously or by otherparenteral routes. In a preferred embodiment, the antibody or fragmentthereof is administered in a dosage of 20 to 2000 milligrams protein perdose. Moreover, the administration may be by continuous infusion or bysingle or multiple boluses. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of immunoconjugate,antibody fusion protein or naked antibody that is in the range of fromabout 1 mg/kg to 20 mg/kg as a single intravenous or infusion, althougha lower or higher dosage also may be administered as circumstancesdictate. This dosage may be repeated as needed, for example, once perweek for four to ten weeks, preferably once per week for eight weeks,and more preferably, once per week for four weeks. It may also be givenless frequently, such as every other week for several months, or morefrequently, such as two- or three-time weekly. The dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one antibody, antigen binding fragment or fusionprotein as described herein. If the composition containing componentsfor administration is not formulated for delivery via the alimentarycanal, such as by oral delivery, a device capable of delivering the kitcomponents through some other route may be included. One type of device,for applications such as parenteral delivery, is a syringe that is usedto inject the composition into the body of a subject. Inhalation devicesmay also be used. In certain embodiments, an anti-pancreatic cancerantibody or antigen binding fragment thereof may be provided in the formof a prefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation of antibody (e.g., Kivitz et al.,Clin. Ther. 2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims. The examplesdiscuss studies employing an exemplary anti-pancreatic cancer monoclonalantibody (e.g., PAM4). Clinical studies with the PAM4 MAb have shownthat a majority of pancreatic cancer lesions were targeted in patientsand there was no indication of uptake in normal tissues. Dosimetryindicated that it was possible to deliver 10 to 20 cGy/mCi to tumors,with a tumor to red marrow dose ratio of 3:1 to 10:1. The data show thatPAM4 is useful for the treatment of pancreatic cancer.

Example 1 Epitope of MUC5AC that Binds to hPAM4 Antibody

PAM4 is a murine monoclonal antibody showing high specificity forpancreatic ductal adenocarcinoma (PDAC) compared with normal tissues andother cancers. Humanized PAM4 labeled with ⁹⁰Y in combination withlow-dose gemcitabine has shown promising therapeutic activity inpatients with metastatic PDAC, and is being evaluated in a phase IIIregistration trial. Prior efforts have suggested the mucin speciesrecognized by PAM4 is human MUC5AC, a secretory mucin expressed de novoin early pancreatic intraepithelial neoplasia and retained throughoutdisease progression. In the present study, we provide further evidencevalidating MUC5AC as the PAM4 antigen, and locate the PAM4-reactiveepitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thusdifferentiating PAM4 from anti-MUC5AC antibodies known to-date.Specifically, we show (i) PAM4-antigen and MUC5AC were co-localized inthe immunocytochemical analysis of multiple human cancer cell lines,including Capan-1, BxPC-3, HT-29, and MCF-7; (ii) MUC5AC-specific siRNAprominently reduced the expression of both MUC5AC and PAM4-antigen inCFPAC-1 cells; (iii) ELISA performed on Capan-1 culture supernatantsfollowing SEPHAROSE®-CL2B chromatography depicted the preferentialbinding of PAM4 to the void-volume fractions, which were furtherrevealed by agarose gel electrophoresis and Western blot to display theladder pattern characteristic of oligomeric MUC5AC; and (iv) by testingthe reactivity of PAM4 with a panel of recombinant fragments of MUC5AC,we demonstrated the N-terminal region comprising Cys2 is essential forbinding to PAM4.

Materials and Methods

Antibodies and Reagents—

Humanized PAM4 (hPAM4) was provided by Immunomedics, Inc. Horseradishperoxidase (HRP)-hPAM4 conjugate was generated using the SureLINK HRPConjugation Kit (Kirkegaard & Perry Laboratories). MAN-5ACI, a rabbitantiserum against MUC5AC (Thornton et al., 1996, Biochem J 316:967-75)was a generous gift from Dr. David J. Thornton (University ofManchester). Commercially available antibodies acquired include thefollowing: four mouse mAbs against MUC5AC (45M1, 2-11M1, 2-12M1, and1-13M1) from Thermo Fisher Scientific, one mouse monoclonal (2Q445) andone rabbit polyclonal (H-160) antibodies against MUC5AC from Santa CruzBiotechnology, one mouse mAb against human MUC1 (MAB6298, hereafterreferred to as α-MUC1) from R&D Systems, one rabbit polyclonal antibodyagainst full-length GFP (α-GFP) from Clontech Laboratories, one rabbitpolyclonal Myc-tag antibody (α-Myc) from Cell Signaling Technology, oneFITC-labeled goat anti-human IgG (FITC-GAH) from Jackson ImmunoResearchLaboratories, and one Cy3-labeled goat anti-mouse IgG (Cy3-GAM) from EMDMillipore. The MUC5AC double-strand siRNA targeting sequence5′-GGAGCCTGATCATCCAGCA-3′ (SEQ ID NO:54) was synthesized by GenScript.SEPHAROSE®® CL-2B was purchased from Sigma-Aldrich.

Cell Culture—

All cell lines were obtained from the American Type Culture Collection(ATCC) and have been authenticated by Promega using Short Tandem Repeat(STR) analysis. BxPC-3, HT-29, LS174T, MCF-7, and Calu-3 were grown inRPMI 1640 medium (Life Technologies) with 10% fetal bovine serum (FBS,Thermo Scientific HyClone); Capan-1 was grown in RPMI 1640 medium with20% FBS; CFPAC-1 was grown in ATCC-formulated Iscove's ModifiedDulbecco's Medium (IMDM) with 10% FBS; SW1990 was grown inATCC-formulated Leibovitz's L-15 Medium with 10% FBS; and PANC-1 wasgrown in Dulbecco's Modified Eagle Medium (Life technologies) plus 10%FBS. All cell lines were incubated at 37° C. in 5% CO₂ except SW1990,which was cultured in 100% air.

Immunocytochemistry—

Cells were plated on 8-chamber slides (Thermo Fisher Scientific) atapproximately 2×10⁴ cells/chamber and incubated overnight at 37° C.Following removal of the medium, cells were fixed in 4% formalin(Sigma-Aldrich) for 15 min at RT, and then treated with 0.1% TritonX-100 in PBS for another 15 min. After washing twice with PBS, cellswere incubated with 10 μg/ml of either hPAM4 or a murine mAb againstMUC5AC or MUC1 in PBS plus 1% BSA for 45 min at RT. Afterwards, cellswere washed twice and incubated with a mixture of FITC-GAH and Cy3-GAMin PBS plus 1% BSA for 30 min at RT. After three washes, chambers weredissembled. Slides were mounted with an antifade solution (VectaShield,Vector Laboratories) containing the nuclear counterstain,4,6-diamidino-2-phenylindole (DAPI). Image acquisition and analyses wereperformed using an Olympus fluorescence microscope with a Kodak camerasystem.

RNA Interference—

CFPAC-1 cells grown to 90% confluence were used for transfection. MUC5ACSiRNA or PBS alone (Mock) was 1:100 diluted into Opti-MEM I Medium (LifeTechnologies) prior to the addition of 1/100 volume of LIPOFECTAMINE®RNAiMAX Reagent (Life Technologies). After 20 min incubation at RT, thesiRNA or Mock mixture was dispersed onto 8-chamber slides (80d/chamber). Meanwhile, cells were trypsinized, washed, diluted incomplete growth medium, and then added at 8×10³ cells/400 μl/chamber.The final RNA concentration was 15.6 nM in a total volume of 480 μl.After 48-h incubation, cells were stained with hPAM4 and anti-MUC5ACmAbs and examined under fluorescence microscope as described above.

Gel Chromatography of Cell Culture Supernatant—

Capan-1 cells were cultured for 3-4 days to reach over 90% confluence.The spent media were collected, mixed with an equal volume of 8 Mguanidine hydrochloride (GdmCl) in 20 mM sodium phosphate buffer (pH 7),and 10-fold concentrated using the Amicon ultrafiltration membrane with30 kDa normal molecular weight limit (EMD Millipore). Gel chromatographywas performed on a SEPHAROSE® CL-2B column (78 cm×2.6 cm) using 4 MGdmCl as the eluent and a flow rate of 40 ml/h. Fractions of 8 mL werecollected and each analyzed for reactivity with hPAM4 and α-MUC-1 byELISA as follows. Briefly, MaxiSorp 96-well plates (Nunc, Roskilde,Denmark) were coated with CL-2B-eluted fractions (100 μl/well) at 37° C.overnight, washed twice with PBS, and blocked with Casein BlockingBuffers (Thermo Fisher Scientific) for 1 h. HRP-hPAM4 or α-MUC1 wasdiluted in PBS and added at 100 l/well. After 1-h incubation at RT,plates with α-MUC1 were washed and incubated further with HRP-GAM for 1h. Plates were washed and bound HRP-hPAM4 or HRP-GAM was detected witho-phenylenediamine dihydrochloride (0.4 mg/ml) in PBS plus 0.03%hydrogen peroxide as a substrate. The optical density was read at 490 nmusing the EnVision 2100 Multilabel Reader (PerkinElmer). The fractionseluted in the void-volume peak were also pooled, dialyzed against thePBS-AG buffer (35.2 mM Na₂PO₄.7H₂O; 0.4 M NaCl; 6.5 mM NaH₂PO₄.H₂O; 150mM arginine; 150 mM monosodium glutamate, pH 8.0), and concentrated with30 kDa Amicon Ultra centrifugal filters (EMD Millipore) for furtheranalysis.

MUC5AC Sandwich ELISA—

MaxiSorp 96-well plates were coated with 100 μl of 2-11M1 (20 μg/ml) inPBS and incubated at 4° C. overnight. After blocking with casein buffer,a 5-fold concentrated void-volume peak pooled from the CL-2Bfractionation of Capan-1 supernatant (hereafter referred to as theCapan-1 void-volume peak) was 2-fold serially diluted and added to theplate at 100 μl/well. After overnight incubation at RT, plates werewashed and detected by HRP-PAM4, or by Biotin-45M1 plus HRP-streptavidinas a positive control.

Agarose Gel Electrophoresis—

Agarose gel electrophoresis was performed as described (Sheehan et al.,2000, Biochem J 347:37-44), with modifications. Briefly, the Capan-1void-volume peak was concentrated in PB S-AG buffer and diluted with gelrunning buffer (40 mM Tris-acetate/1 mM EDTA, 0.1% SDS, pH 8.0). Inselective experiments, serum samples from normal subjects or pancreaticcancer patients were mixed with an equal volume of 8 M guanidinehydrochloride (GdmCl) and dialyzed into gel running buffer. All sampleswere supplemented with 1 M urea, 3% glycerol and 0.02% bromophenol bluebefore loading into thin wells shaped with a 0.8 mm-thick comb in 0.7%agarose gel (5.7 cm×8.3 cm). Electrophoresis was performed at 30 V for 4to 8 h in the Horizon 58 Electrophoresis Apparatus (LABRepCo).

Construction of Expression Vectors for MUC5AC Recombinant Fragments—

The pSM-MUC5AC-CH-long expression vector (Lidell et al., 2008, FEBS J275:481-9; Lidell & Hansson, 2006, Biochem J 399: 121-9), which encodesa signal sequence, a Myc tag (EQKLISEEDL, SEQ ID NO:55), the humanMUC5AC (Swiss-Prot accession no. P98088) C-terminal cysteine-rich part(AA3993-5030), and a histidine tag, was kindly provided by Dr. GunnerHansson of Gothenburg University (Gothenburg, Sweden). Additionalvectors were constructed from pSM-MUC5AC-CH-long by replacing the DNAsequence of AA3993-5030 with that of AA1-1217, AA1218-2199, AA1218-1517,AA1575-2052, AA1725-2052, AA1575-1723/1903-2052, AA1575-1853, andAA1575-1725, to express D1-D2-D′-D3 (b-fragment), 11P15-Cys1-2-3-4-5(c-fragment), 11P15-Cys1 (d-fragment), Cys2-3-4 (e-fragment), Cys3-4(f-fragment), Cys2/4 (g-fragment), Cys2-3 (h-fragment), andCys2+(i-fragment), respectively, as listed in Table 2. In addition, fourGFP-fused fragments were produced by replacing the Myc tag with a fullGFP sequence in the vectors encoding Cys2-3-4, Cys3-4, Cys2/4, andCys2-3, resulting in the e*-, f*-, g*- and h*-fragment, respectively.Myc-tagged Cys2-3-4 and Cys2+ were also expressed in E. coli, andpurified from the inclusion body using HIS-Select Nickel Affinity Gel(Sigma-Aldrich), and refolded.

TABLE 2 Recombinant MUC5AC fragments MUC5AC MW Expression Fragment TagDomains AA # (P98088) ^(a)(Da) PANC-1 E. coli a Myc Cys9-D4-B-C-CK3992-5030 116,140 + b Myc D1-D2-D′-D3   1-1217 136,727 + c Myc11P15-Cys1-2-3-4-5 1218-2199 109,380 + d Myc 11P15-Cys1 1218-151735,704 + e Myc Cys2-3-4 1575-2052 56,444 + + f Myc Cys3-4 1725-205239,686 + g Myc Cys2/4 1575-1725/1903-2052 37,171 + h Myc Cys2-31575-1853 35,452 + I Myc Cys2+ 1575-1725 20,504 + e* GFP Cys2-3-41575-2052 81,742 + f* GFP Cys3-4 1725-2052 64,983 g* GFP Cys2/41575-1725/1903-2052 62,468 + h* GFP Cys2-3 1575-1853 60,749 +

Transient Expression of Recombinant MUC5AC Fragments—

One day prior to transfection, PANC-1 cells were seeded in a 24-wellplate at 2×10⁵/well and held at 37° C. overnight. Transfection wasperformed using Lipofectamine 2000 (Life Technologies) with and withoutthe recombinant plasmid DNA of interest. After 72 h, the spent mediawere collected and analyzed by Western blot following gelelectrophoresis.

Western Blot—

Samples were electrophoresed in the same gel or different gels under thesame conditions. After electrophoresis, samples were transferred (100V,1 h) onto a nitrocellulose membrane using the Mini TRANS-BLOT® cellsystem (Bio-Rad Laboratories) and probed with hPAM4, an anti-MUC5ACantibody, α-GFP, or α-Myc, as indicated. The signals were developed withSUPERSIGNAL™ West Dura Chemiluminescent Substrate (Thermo FisherScientific).

Results

Co-Localization of the hPAM4 Antigen and MUC5AC in Different Cell Lines—

Several cell lines were subjected to cytofluorometry in order toevaluate localization patterns (heterogeneous and/or homogenous) ofMUC1, MUC5AC, and/or MUC17, as detected by hPAM4 and othermucin-specific mAbs. The cell lines examined included those derived fromhuman pancreatic (CaPan-1, BxPC3, CFPAC-1, and AsPC-1), colorectal(HT-29 and LS174 T), breast (MCF-7), and lung (A549) carcinomas. Asshown in FIG. 1, in each of the cell lines examined, hPAM4 exclusivelyco-localized with MUC5AC (as identified by two anti-MUC5AC mAbs, 2-11M1and 2-12M1), but not with MUC1 or MUC17 (data not shown), suggestingthat MUC5AC is the hPAM4-reactive antigen.

Co-Knockdown of the hPAM4 Antigen and MUC5AC by MUC5AC-Specific siRNA—

The disparate localization between PAM4 and anti-MUC1 or anti-MUC17indicates that PAM4 reacts with neither MUC1 nor MUC17. On the otherhand, the co-localization of PAM4 and the two anti-MUC5AC mAbs (2-11M1and 2-12M1) is consistent with PAM4 being specific for MUC5AC (Gold etal., 2013, Mol Cancer 12:143). To investigate if hPAM4 associates withMUC5AC, we employed the RNAi method to specifically knockdown MUC5AC. Asshown in the upper panel of FIG. 2, hPAM4 and 2-11M1 are co-localized inuntreated CFPAC-1 cells, as well as the mock-treated (transfection agentalone) cells. In contrast, treatment with MUC5AC-specific siRNA resultedin substantially reduced immunostaining for both 2-11M1 and hPAM4.Moreover, as shown in the bottom panel of FIG. 2, siRNA knockdown ofMUC5AC did not alter the anti-MUC1 immunostaining, providing furtherevidence that hPAM4 is not reactive with MUC1.

Presence of the hPAM4 Antigen in the Culture Supernatant ofMucin-Producing Carcinoma Cell Lines—

MUC5AC is a highly oligomeric secretory mucin that has been isolatedfrom cell culture and in vivo mucous secretions (Sheehan et al., 2000,Biochem J 347:37-44; Hovenberg et al., 1996, Glycoconj J 13:839-47). Ourearly studies showed that hPAM4 reacts with mucin derived from theCaPan-1 xenografted human PDAC (Gold et al., 1994, Int J Cancer57:204-10). In the current study, we used SEPHAROSE® CL-2B molecularsieve chromatography to separate the mucin species secreted into thesupernatant of CaPan-1. The eluted fractions were then examined forimmunoreactivity with hPAM4 and α-MUC1. As shown in FIG. 3A,PAM4-reactive substance was present predominantly in the void-volumepeak, whereas only subsequently eluted fractions were found reactivewith α-MUC1. When the Capan-1 void-volume peak was probed withanti-MUC5AC antibodies, we found a positive response with 45M1, 1-13M1,and H-160, but not 2Q445, as shown in FIG. 3B. It is noted that thevoid-volume peaks obtained from other cancer cell lines known to secretMUC5AC, such as HT-29 (Sheehan et al., 2000, Biochem J 347:37-44), LS174T (Asker et al., 1998, Biochem J 335:381-7), SW1990 (Hoshi et al,2011, Int J Oncol 38:619-27), CFPAC-1 (Luka et al., 2011, J BiomedBiotechnol 2011:93475728), and Calu-3 (Rose et al., 2000, J Aerosol Med13:245-61), all tested positive for reactivity with hPAM4 (data notshown).

Direct evidence that correlates the hPAM4-reactive substance in theCapan-1 void-volume peak with MUC5AC is provided by a sandwich ELISAformatted to quantify the MUC5AC captured by 2-11M1, which reacts withthe N-terminal domains of MUC5AC (Nollet et al., 2004, HybridHybridomics 23:93-930). As shown in FIG. 3C, 2-11M1-captured MUC5ACcould be detected by hPAM4 in a dose-dependent manner, demonstratingthat hPAM4 binds to a different region of MUC5AC from 2-11M1. Theadditional results obtained with 45M1, which serves as a positivecontrol, also support the previous conclusions that the epitopes of 45M1(Lidell et al., 2008, FEBS J 275:481-9) and 2-11M1 on MUC5AC arenon-overlapping.

Electrophoretic Resolution of the hPAM4-Reactive Void-Volume Fractionson Agarose Gel—

To further verify that the hPAM4-reactive substance is MUC5AC, theCapan-1 void-volume peak was separated by electrophoresis on 0.7%agarose gel, and subsequently probed with hPAM4 (FIG. 4A, left panel),45M1 (FIG. 4A, middle panel), or MAN-5ACI (FIG. 4A, right panel) byWestern blotting. Under non-reducing conditions, a group of bandsresembling the ladder-like pattern reported for MUC5AC (Sheehan et al.,2000, Biochem J 347:37-44, Sheehan et al., 2004, J Biol Chem279:15698-705) was clearly discerned by all three antibodies in the samegel. In contrast, under reducing conditions, two bands were revealed byMAN-5ACI, but undetectable by either hPAM4 or 45M1, which corroboratethe previous findings that the predominant fast-migrating band and theminor band trailing behind represent the MUC5AC monomer and areduction-resistant dimer, respectively (Sheehan et al., 2000, Biochem J347:37-44), and that neither 45M1 (Lidell et al., 2008, FEBS J275:481-9) nor hPAM4 (Gold et al., 1994, Int J Cancer 57:204-10) shouldreact with a reduced mucin.

Detection of hPAM4-Reactive Substance in Serum Samples of PancreaticCancer Patients—

The visualization of MUC5AC by hPAM4 as a characteristic ladder inWestern blot following agarose gel electrophoresis prompted us toexamine whether such a pattern could be demonstrated for patient serumfound positive with the presently formulated PAM4-based assay (Picozziet al., 2014, J Clin Oncol 132:4026; Gold et al., 2010, Cancer EpidemiolBiomarkers Prev 19:2786-94). As shown in FIG. 4B, a broad band migratingfaster than MUC5AC monomer was detected by hPAM4 and severalMUC5AC-specific antibodies, such as 2-11M and H-160, but not by 45M1,suggesting the PAM4-reactive antigen in patient serum could be derivedfrom an immature MUC5AC variant, or a breakdown product of matureMUC5AC.

Mapping of the hPAM4 Epitope on MUC5AC—

The disparity in the reactivity of hPAM4 and 2Q445 with the Capan-1void-volume peak, as noted in FIG. 3B, suggests that the hPAM4 epitopeis not in the tandem repeat region of MUC5AC recognized by 2Q445(Perez-Vilar et al., 2006, J Biol Chem 281:4844-55). Therefore, weexcluded the tandem repeat region (AA2199-3992) and decided to expressin PANC-1 cells three large recombinant fragments (designated as a, b,and c) that comprise the remainder of MUC5AC (FIG. 5A). We found thathPAM4 did not react with the C-terminal a-fragment (AA3992-5030) or theN-terminal b-fragment (AA1-1217), suggesting its epitope was locatedoutside the N-terminal D1-D2-D′-D3 domains and the C-terminal regionencompassing Cys9-D4-B-C-CK domains. In contrast, the c-fragment(AA1218-2199), which spans the five N-terminal cysteine-rich subdomains(Cys1-2-3-4-5), reacted with hPAM4 as shown by Western blot (FIG. 5B,left panel). Expectedly, the c-fragment was found to react also with1-13M1 (data not shown) and 45M1 (FIG. 5B, right panel), which recognizecysteine-rich subdomains of class-2 (Cys2 and Cys4) and class-3 (Cys3,5, 6, 7 and 9), respectively. We next expressed two sub-fragments (d ande) within the c-fragment and showed (FIG. 5C, left panel) that hPAM4failed to react with the d-fragment (AA1218-1517) comprising 11P15-Cys1,but strongly stained the e-fragment (AA1575-2052) comprising Cys2-3-4.We then expressed three overlapping sub-fragments (f, g, and h) of thee-fragment and showed (FIG. 5D, left panel) hPAM4 stained the g-fragment(AA1575-1725 joined to AA1903-2052, comprising Cys2 and Cys4 with Cys3deleted), but barely the f-fragment (AA1725-2052, comprising Cys3-4) orthe h-fragment (AA1575-1853, comprising Cys2-3). The differentialreactivity of hPAM4 observed for the e-, f-, g-, and h-fragments wasconfirmed (FIG. 5E, left panel) with the respective GFP-fusedcounterparts (the e*-, f*-, g*- and h*-fragments); the expression ofeach was clearly shown by Western blot with anti-GFP (FIG. 5E, rightpanel). Together, these results indicate that (i) the hPAM4 epitoperesides within the e-fragment, which contains the Cys2-3-4 region; (ii)the presence of Cys2 or Cys4, or both, is needed for recognition byhPAM4; (iii) Cys3 is essential for the binding of 45M1, since it stainedeach of the c- (FIG. 5B, right panel; FIG. 5C, rightmost panel), e-, f-,and h-fragments (FIG. 5C, rightmost panel; FIG. 5D, right panel), all ofwhich contains Cys3; but not the g-fragment (FIG. 5D, right panel),which lacks Cys3; and (iv) the validity of the d-fragment was supportedby its positive staining with H-160 (FIG. 5C, middle panel), whoseepitope was reported to reside in AA1214-1373 (33) contained in thed-fragment.

The successful expression of Cys2-3-4 (AA1575-2052) and Cys2+(AA1575-1725) in E. coli, as evidenced by the coomassie blue staining(FIG. 6A) and Western blot using anti-Myc (FIG. 6B), was instrumental infurther defining the location of the hPAM4 epitope to the Cys2subdomain. The unglycosylated Cys2-3-4 and Cys2+ were isolatedpredominantly as monomeric species of 55.4 and 20.5 kDa, respectively.As shown in FIG. 6C, hPAM4 reacts with non-reduced, but not the reduced,Cys2-3-4 and Cys2+. Although 1-13M1 also targets Cys2 or Cys4, itsbinding to both non-reduced and reduced Cys2+ (FIG. 6D) differentiatesit from hPAM4. Thus, we further establish that the hPAM4 epitope, beingreduction-sensitive, is conformational, located within the Cys2subdomain, and unlikely involving carbohydrates. We speculate that theweakly positive bands observed for hPAM4 in lanes 3 and 4 of FIG. 6Ccould result from reformation of the disulfide bond to a varying degreein the process of blotting, which would restore the hPAM4 epitope.

Discussion

In the past decade, concerted efforts in the search of biomarkers forPDAC have produced compelling evidence that mucins are aberrantlyexpressed in this devastating malignancy, and have diverse biologicalfunctions in tumor development, progression, metastasis, and drugresistance (Kaur et al., 2013, Nat Rev Gastroenterol Hepatol 10:607-20).Moreover, a number of studies (Kaur et al., 2013, Nat Rev GastroenterolHepatol 10:607-20; Lau et al., 2004, Am J Clin Pathol 122:61-9; Remmerset al., 2013, Clin Cancer Res 19:1981-93) have shown that bothcell-tethered and secreted mucins display different expression profilesin pancreatic cancer when compared to normal pancreas. As a de novomucin in pancreatic cancer, MUC5AC could be detected as early as thepre-malignant/dysplastic stages (Nagata et al., 2007, J HepatobiliaryPancreat Surg 14:243-54), and was identified in a high percentage ofPDAC (Remmers et al., 2013, Clin Cancer Res 19:1981-93; Yamazoe et al.,2011, Pancreas 40:896-904; Kanno et al., 2006, Pancreas 33:391-6).

Our own endeavors for over 20 years have focused on the exploration ofmucin-reactive PAM4 as a potential diagnostic and therapeutic agent forPDAC. Although we have recently proposed MUC5AC to be the PAM4 antigen(Gold et al., 2013, Mol Cancer 12:143), the identification of the PAM4epitope has lagged behind its clinical development, mainly due to thechallenges encountered in characterizing MUC5AC, which is polymeric,heavily O-glycosylated, and present in several variant forms (Thorntonet al., 2008, Annu Rev Physiol 70:459-86; Silverman et al., 2001,Glycobiology 2001; 11:459-71; Guo et al., 2014, Am J Respir Cell MolBiol 50:223-32).

In the current Example, we provide additional evidence fromimmunocytochemistry, RNA interference, and biochemical studies thatauthenticates MUC5AC as the hPAM4 antigen; and more importantly, havelocated the PAM epitope to the N-terminal region comprising Cys2 throughthe recombinant expression of MUC5AC domains (Backstrom et al., 2013,Mol Biotechnol 54:250-6). We should note that DEGYTFCESPR (SEQ IDNO:56), one of the 6 MUC5AC peptides most frequently detected in thepancreatic cystic lesions with malignant potential, and not in thebenign lesions, is located in the Cys2 and Cys4 subdomains, as reportedin a very recent study of mucin proteomics (Jabbar et al., 2014, J NatlCancer Inst 106:djt439).

Based on their sequence similarity (Escande et al., 2001, Biochem J358:763-72), the 9 Cys subdomains of MUC5AC have been characterized (Guoet al., 2014, Am J Respir Cell Mol Biol 50:223-32) as Class I (Cys1),Class II (Cys2, Cys4; 98% identical), and Class III (Cys3, Cys5-9; 96%identical). Whereas each subdomain contains about 110 amino acidresidues, including 10 remarkably conserved cysteine residues involvedin intramolecular disulfide bonds, there is only one potentialO-glycosylation site and no potential N-glycosylation site. Thesestructural features appear to match the characteristics of the hPAM4epitope. Earlier work (Gold et al., Int J Cancer 1994, 57:204-10) showedthat the reactivity between PAM4 and its mucin antigen was negativelyaffected by heating, reduction of disulfide bonds, or certain proteasedigestion, suggesting that the PAM4 epitope is a conformationalglycopeptide. While we have confirmed that reduced MUC5AC no longerreacts with hPAM4, the results obtained from the unglycosylated Cys2-3-4and Cys2+ of this study also indicate that the hPAM epitope is retainedunder denaturing conditions (or can be readily restored followingblotting or immobilization and washing), and unlikely to involvecarbohydrates. Because Cys2 and Cys4 are 98% identical in amino acidsequence, including all of the 10 conserved cysteine residues, we expectthe hPAM4 epitope is present on Cys4 also.

It is worthy of note that among the various anti-MUC5AC antibodies withmapped epitopes, which include the mouse mAbs of the M1 series: 1-13M1(Rose et al., 2000, J Aerosol Med 13:245-61), 2-11M1 (Rose et al.,2000), 9-13M1 (Rose et al., 2000), 19M1 (Rose et al., 2000), 21M1 (Roseet al., 2000), 62M1 (Rose et al., 2000), 45M1 (Sheehan et al., 2000,Biochem J 347:37-44), and 2-12M1 (Sheehan et al., 2000); other murinemAbs such as CLH2 (Reis et al., 1997, Int J Cancer 74:112-21), SOMU1(Rose et al., 2000, J Aerosol Med 13:245-61), 2Q445 (Sheehan et al.,2004, J Biol Chem 279:15698-705), and NPC-1C (U.S. Pat. No. 7,763,720);and two rabbit polyclonal antibodies, H-160 (Perez-Vilar et al., 2006, JBiol Chem 281:4844-55) and MAN-5ACI (Thornton et al., 1996, Biochem J316:967-75), 1-13M1 is the only mAb reported to react with Cys2/4subdomains of MUC5AC. Our data, however, indicate that 1-13M1 binds to areduction-insensitive epitope, thus being different from that of hPAM4.

Because the Cys2, Cys3 and Cys4 subdomains are flanked bythreonine/serine/proline (TSP)-rich sequences, which contain numerousO-glycosylation sites, we further note that the accessibility of hPAM4to its epitope on Cys2 (or Cys4) could be masked by the surroundingoligosaccharides either structurally or in a conformation-dependentmanner, or both. Accordingly, hPAM4 would prevail for underglycosylatedMUC5AC, whose expression in epithelial cancers in general, and PDAC inparticular, including the early-stage pancreatic cancer precursors, hasnot been as well studied as that of underglycosylated MUC1 (Reis et al.,1998, Int J Cancer 79:402-10).

In conclusion, we have located the hPAM4 antigen to the N-terminal Cys2of MUC5AC and characterized it as a reduction-sensitive,carbohydrate-free epitope, whose access may be restricted by thesurrounding oligosaccharides in the flanking TSP-domains. We believe theultimate delineation of the hPAM4 epitope may lead to its exploration asa candidate for vaccine development, while providing valuable insightfor diagnosis and treatment of MUC5AC-expressing cancers, such asbiliary tract cancer (Wongkham et al., 2003, Cancer Lett 195:93-9),colorectal cancer (Bu et al., 2010, World J Gastroenterol 16:4089-94),and gastric cancer (Wang et al., 2003, J Surg Oncol 83:453-60), inaddition to PDAC.

Example 2 Therapeutic Dosages of ⁹⁰Y-Labeled Anti-MUC5AC Antibody forHuman Pancreatic Cancer

For patients with metastatic pancreatic adenocarcinoma, there are noapproved or established treatments beyond 2^(nd) line. A study offractionated radioimmunotherapy was undertaken, administering⁹⁰Y-clivatuzumab tetraxetan (yttrium-90-radiolabeled hPAM4 anti-MUC5ACantibody) with or without low radiosensitizing doses of gemcitabine.

Methods:

Fifty-eight patients with 3 median (range 2-7) prior treatments weretreated on Arm A (N=29, ⁹⁰Y-clivatuzumab tetraxetan, weekly 6.5 mCi/m²doses×3, plus gemcitabine, weekly 200 mg/m² doses×4 starting one weekearlier) or Arm B (N=29, ⁹⁰Y-clivatuzumab tetraxetan alone, weekly 6.5mCi/m² doses×3), repeating cycles after 4-week delays. Safety andefficacy were evaluated.

Results:

Cytopenias (predominantly transient thrombocytopenia) were the onlysignificant toxicities. Fifty-three patients (27 Arm A, 26 Arm B, 91%overall) completed ≧1 full treatment cycles, with 23 (12 Arm A, 11 ArmB; 40%) receiving multiple cycles, including 7 (6 Arm A, 1 Arm B; 12%)given 3-9 cycles. Two patients in Arm A had partial responses by RECISTcriteria. Kaplan-Meier overall survival (OS) appeared improved in Arm Av B (hazard ratio [HR] 0.55, 95% CI: 0.29-0.86; P=0.017, log-rank) andthe median OS for Arm A v Arm B increased to 7.9 v 3.4 months withmultiple cycles (HR 0.32, P=0.004), including 3 patients in Arm Asurviving >1 year.

Conclusions:

With these surprising results, clinical use of ⁹⁰Y-clivatuzumabtetraxetan and low-dose gemcitabine is feasible in metastatic pancreaticcancer patients beyond 2^(nd) line. A Phase III trial is now underway inthis setting.

Introduction

The outlook for patients with advanced pancreatic adenocarcinoma remainspoor (Hidalgo, 2010, N Engl J Med 362:1605-17). In the frontline, mediansurvival was 6.2-6.7 months with gemcitabine alone (Burris et al., 1997,J Clin Oncol 15:2403-13) or with erlotinib (Moore et al., 2007, J ClinOncol 25:1960-6), 8.5 months combined with albumin-bound paclitaxel (VonHoff et al., 2013, N Engl J Med 369:1691-1703), and 11.1 months forthose able to tolerate combination chemotherapy (FOLFIRINOX) (Conroy etal., 2011, N Engl J Med 364:1817-25). Beyond 1st line, the survivaladvantage with chemotherapy remains limited (Rahma et al., 2013, AnnOncol 24:1972-9; Oettle et al., 2014, J Clin Oncol 32:2423-9) and aftertwo prior treatments (one usually gemcitabine-based, the otherfluoropyrimidine-based), there are no accepted treatments (Seufferleinet al., 2012, Ann Oncol 23(suppl 7):vii33-40; Almhanna & Kim, 2008,Oncology (Williston Park) 22:1176-83).

We pursued radioimmunotherapy to target and directly irradiate tumorsites without needing to physically overcome transport barriers inpancreatic cancer (high interstitial pressure, dense stromal reaction)or be incorporated into the tumor cells to be effective (Koay et al.,2014, J Clin Invest 124:1525-36). PAM4, an anti-MUC5AC monoclonalantibody selectively binding to pancreatic adenocarcinoma mucin, provedactive when radiolabeled in preclinical models of human pancreaticcancer (Cardillo et al., 2001, Clin Cancer Res 7:3186-92; Gold et al.,1997, Int J Cancer 71:660-7). However, dosage studies in animal modelsystems are generally not predictive of effective therapeutic dosages inhumans (Reagan-Shaw et al., 2007, FASEB J 22:659-61), requiring clinicalstudies in human subjects to establish safe and effective therapeuticdosages.

After humanization and conjugation with DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid), thechelate-hPAM4 conjugate (clivatuzumab tetraxetan) was labeled with90-yttrium (⁹⁰Y), a beta-emitting radionuclide with a radiationpath-length of ˜5 mm suitable for bulky tumors. ⁹⁰Y-clivatuzumabtetraxetan was initially administered as a single dose (Gulec et al.,2011, Clin Cancer Res 17:4091-100), but fractionated doses should bemore effective (DeNardo et al., 2002, Cancer 94:1332-48). Gemcitabine isa known radiosensitizer (Morgan et al., 2008, Clin Cancer Res14:6744-50), tolerated clinically at low doses with externalradiotherapy (Pauwels et al., 2005, Oncologist 10:34-51), andpreclinical studies showed enhanced anti-tumor activity combining⁹⁰Y-labeled PAM4 with gemcitabine (Cardillo et al., 2002, Int J Cancer97:386-92; Gold et al., 2003, Clin Cancer Res 9:3929S-37S; Gold et al.,2004, Int J Cancer 109:618-26).

In the frontline, fractionated doses of ⁹⁰Y-clivatuzumab tetraxetancombined with 200 mg/m² doses of gemcitabine achieved 11.8 months mediansurvival for those patients given repeated treatment cycles; manageablemyelosuppression was the principal side-effect (Ocean et al., 2012,Cancer 118:5497-505). There is an unmet medical need for further therapyin pancreatic cancer patients who have received and shown resistance toor relapsed from two or more prior therapies. Radioimmunotherapy may beparticularly attractive for patients considering continued treatment,but unable or unwilling to tolerate the side effects of furtherchemotherapy.

Methods

This Example reports the results of an open-label, multicenter phase Ibstudy of ⁹⁰Y-clivatuzumab tetraxetan administered with or without 200mg/m² gemcitabine in patients with metastatic pancreatic adenocarcinomaafter ≧2 prior therapies. Primary study objectives included evaluatingtreatment safety and tolerability in this setting. Additional objectiveswere to obtain evidence of efficacy based on survival, CT imaging andCA19-9 serum levels, assess the contribution of gemcitabine to thistreatment regimen, and evaluate any immunogenicity toward thisantibody-based regimen.

Population.

Adults ≧18 years old with metastatic pancreatic adenocarcinoma hadreceived ≧2 prior chemotherapy regimens for their advanced disease andhad measureable disease by CT imaging, but no CNS metastases or bulkydisease (no single mass ≧10 cm). Other requirements included Karnofskyperformance status ≧70%, hemoglobin ≧9 g/dL, neutrophils [ANC]≧1500/mm³,platelets ≧100,000/mm³, creatinine and bilirubin ≧1.5×IULN(institutional upper limit of normal), AST and ALT≧2.0×IULN, with anyprior external radiation therapy <2000 cGy to lungs and kidneys, <3000cGy to liver, <30% of red marrow, and with ≦Grade 2 nausea/vomiting,anorexia, or signs of intestinal obstruction.

Treatment and Assessments.

Immunomedics, Inc., (Morris Plains, N.J.) provided clivatuzumabtetraxetan to local commercial radiopharmacies for ⁹⁰Y-radiolabeling.Based on the prior study (Ocean et al., 2012, Cancer 118:5497-5506), a6.5 mCi/m² dose was selected for this population, with ⁹⁰Y-clivatuzumabtetraxetan administered in the hospital nuclear medicine department byslow injection over 5-10 minutes. Commercially available gemcitabineprepared by the hospital pharmacy was given intravenously over 30minutes.

Patients were alternately assigned to treatment arms, withoutconsideration of prior history other than meeting eligibility criteria.In Arm A, patients received 6.5 mCi/m² ⁹⁰Y-clivatuzumab tetraxetanonce-weekly for 3 weeks together with 200 mg/m² gemcitabine once-weeklyfor 4 weeks starting 5 days before beginning ⁹⁰Y-clivatuzumab tetraxetanand then 2 days after each dose. In Arm B, patients received only 6.5mCi/m² ⁹⁰Y-clivatuzumab tetraxetan once-weekly for 3 weeks. For botharms, treatment cycles were repeated after 4 weeks following the lastdose until unacceptable toxicity, progressive disease, or patientwithdrawal. The full ⁹⁰Y-clivatuzumab tetraxetan dose was administeredfor ANC≧1000/mm³ and platelets ≧100,000/mm³; otherwise, a 75% dose forANC≧750/mm³ and platelets ≧75,000/mm³, or a 50% dose for lower valueswith ANC≧500/mm³ and platelets ≧50,000/mm³, with the dose held ifANC<500/mm³ or platelets <50,000/mm³ and treatment delayed on a weeklybasis until blood levels permitted dosing to continue. Gemcitabine doseswere held if ⁹⁰Y-clivatuzumab tetraxetan doses were held, but wereotherwise given without reduction.

Adverse events were graded by NCI-CTCAE v4.0 (National Cancer Institute(NCI). Common terminology criteria for adverse events (CTCAE) version4.0). Vital signs, physical examination, blood counts, serumchemistries, and CA19-9 serum levels were evaluated during treatmentcycles, then 4, 8, and 12 weeks after last cycle. Serum samples wereevaluated by enzyme immunoassay for any human anti-hPAM4 antibodies(HAHA). CT scans were interpreted by local radiologists every 4 weeksafter each cycle until progression or 12 weeks post-treatment. Tumorlesion changes were categorized as a complete response (CR), partialresponse (PR), stable (SD), or progressive disease (PD) by RECIST v1.0[27]. ¹⁸F-FDG-PET or PET/CT imaging was optional. All patients werefollowed for survival.

Statistical Analysis.

Overall survival (OS) from first dose to death or last contact wasanalyzed by Kaplan-Meier methods. Other results were summarized bydescriptive statistics. To determine if this approach is sufficientlyactive in this population, and if low-dose gemcitabine adds to thetherapeutic activity, a SWOG two-stage design was used with a treatmenttarget of ≧25% disease control (PR or CR, or else SD for at least 8weeks from treatment initiation) versus ≧5% for an inactive therapy,resulting in a sample size of ≧50 patients (25 per arm) for this study.

Results

Patients and Study Treatment.

Fifty-eight patients with a median of 3 (range, 2-7) prior chemotherapyregimens were enrolled. All had received gemcitabine-containing regimens(21% with nab-paclitaxel) while 97% had receivedfluoropyrimidine-containing regimens (50% with FOLFIRINOX). Twenty-ninepatients were treated in both Arm A (gemcitabine) and Arm B (nogemcitabine). Table 3 summarizes patient demographics and baselinecharacteristics for each arm.

TABLE 3 Demographics and baseline characteristics Arm A Arm B Patients,N 29 29 Sex, M/F 19/10 14/15 Age, median (range) 62 (39-73) 66 (51-80)Karnofsky performance status, N 90-100 14 10 70-80 15 19 Hematology,median (range) Platelets (×1000/μL) 211 (100-577) 224 (113-570)Neutrophils (×1000/μL) 5.2 (1.9-15.1) 5.4 (3.0-13.4) Hemoglobin (g/dL)11.3 (9.2-15.1) 11.6 (9.4-15.8) Years from diagnosis, median 1.8(0.3-4.1) 1.5 (0.4-3.7) (range) Stage IV disease, N (%) 29 (100%) 29(100%) Extent of disease, median (range) CA19-9 (U/mL) 863 (0.8-UL*)2720 (6.5-UL*) Sum of index lesions (cm) 9.8 (1.7-19.0) 7.1 (1.2-22.2)Tumor location, N (%) Pancreas, including resection bed 11 (38%) 15(52%) Liver 14 (48%) 17 (59%) Other abdomen sites 13 (45%) 16 (55%) Lung13 (45%) 12 (41%) Prior therapies Pancreatectomy, N (%) 15 (52%) 9 (31%)External radiation, N (%) 10 (34%) 8 (28%) Chemotherapy regimens Median(range) 3 (2-7) 3 (2-6) Most frequent, N (%) Gemcitabine-containing 29(100%) 29 (100%) Gemcitabine/nab-paclitaxel 6 (21%) 6 (21%)Fluoropyrimidine-containing 28 (97%) 28 (97%) FOLFIRINOX^(†) 17 (59%) 12(41%) *UL = upper limit of quantitation, nominally >200,000 U/mL.^(†)Irinotecan, oxaliplatin, and 5-fluorouracil/leucovorin combination.

No drug-related interruptions, discontinuations or adverse reactionsoccurred with treatment administrations. Five patients rapidlydeteriorated before finishing one cycle (stroke, entered hospice,biliary obstruction, pulmonary embolism, severe constipation). Patientsterminated further treatment due to disease progression/clinicaldeterioration or other treatment-unrelated events, with 30/58 (52%)patients (15 per arm) completing only one cycle, and 23 (40%) patients(12 Arm A, 11 Arm B) receiving multiple cycles, including 16 (6 Arm A,10 Arm B) given 2 cycles and 7 (6 Arm A, 1 Arm B) given 3 to 9 cycles.For cycle 1, 8/58 (14%) patients had ≧1 doses of ⁹⁰Y-clivatuzumabtetraxetan reduced to 75%; but, besides the 5 patients who rapidlydeteriorated, no doses were held. For repeated cycles, 16/23 (70%)patients had ≧1 doses reduced to 75% (N=7) or 50% (N=9), including 11(48%) who also had ≧1 doses held.

Adverse Events.

Events considered at least possibly treatment-related werethrombocytopenia, 50% of patients; fatigue, 26%; anemia, 22%; nausea,16%; leukopenia, neutropenia, 12% each; abdominal pain, anorexia,vomiting, diarrhea, 9% each; bleeding, fever, chills, 7% each; dyspnea,hyperbilirubinemia, headache, 5% each; others <5%. These included Grade≧3 events of thrombocytopenia, 19%; anemia, leukopenia, neutropenia, 7%each; others ≦2%.

Comparison of events regardless of assumed treatment relationship showslimited differences between treatment arms, and AEs occurring morefrequently (>10% difference) in arm A (fatigue, neutropenia, leukopenia,nausea, diarrhea, dyspnea, alkaline phosphatase, headache) or arm B(ascites, asthenia, gastrointestinal pain or tenderness) were primarilylimited to Grade 1-2 events (Table 4).

TABLE 4 Patient Incidence of Most Frequent Adverse Events Regardless ofAssumed Relationship to Treatment.* Arm A Arm B All Grade All Adverseevent, % Grades ≧3 Grades Grade ≧3 Laboratories Thrombocytopenia 62 2152 17 Anemia 45 14 38 10 Neutropenia 28 10 3 3 Leukopenia 24 14 3 3Alkaline phosphatase 24 3 10 0 Hyponatremia 21 7 14 7 Aspartateaminotransferase 17 0 7 7 Lymphopenia 14 10 17 10 Hyperbilirubinemia 143 21 3 Hyperglycemia 14 3 14 7 Hypoalbumemia 14 0 3 3 Clinical EventsFatigue 76 3 38 7 Nausea 41 0 24 3 Anorexia 31 0 24 7Abdominal/gastrointestinal 31 3 48 10 pain or tenderness Constipation 280 28 3 Diarrhea 28 0 10 0 Dyspnea 28 7 14 0 Infection 24 7 17 7 Vomiting21 3 28 3 Abdominal distension 17 0 7 0 Bleeding 17 3 14 7 Back Pain 177 14 0 Cough 17 0 7 0 Dehydration 14 3 17 3 Headache 14 0 0 0Hypertension 14 7 3 0 Fever 14 0 14 0 Pleural effusion 10 7 17 7Peripheral edema 10 0 14 0 Ascites 0 0 14 3 Asthenia 0 0 14 3 *Eventsoccurring in >10% of the 29 patients in either treatment arm

Six patients (3 per arm) had serious events considered at least possiblytreatment-related. Two patients had cerebrovascular accidents due tothromboembolic or watershed events, one after the first dose and theother one month after cycle 1, and two other patients developedconsumptive coagulopathies, one after cycle 1 with thrombocytopenia,deep venous thrombosis and sub-acute cerebral infarcts, the other aftercycle 2 with thrombocytopenia, acute renal failure and fatalgastrointestinal hemorrhage. One patient developed fever after cycle 3with negative cultures but responded to antibiotics, while anotherpatient with a history of severe infections developed fatalGram-negative bacteremia during cycle 2; both had undergone recentbiliary stent placements and were not neutropenic at time of event.

Overall, 20/58 (34%) patients (10 per arm) developed Grade ≧3thrombocytopenia (4 given platelets), 14 (11 Arm A, 3 Arm B; 24%overall) developed Grade ≧3 neutropenia (4 given cytokine support), and11 (7 Arm A, 4 Arm B; 19% overall) developed Grade ≧3 anemia (8transfused). In patients with follow-up data, only 4 events (allthrombocytopenia) remained at Grade 4 levels >7 days, while all Grade 3cytopenias recovered to Grade 2 levels within 12 weeks. Grade ≧3cytopenias generally increased with repeated cycles but Grade 4occurrences generally remaining limited (Table 5).

TABLE 5 Grade 3 and 4 Hematological Toxicity by Treatment Cycle Thrombo-cytopenia Neutropenia Anemia Grade Grade Grade Grade Grade Grade Cycle N3 4 3 4 3 4 Cycle 58 4 (7%)  6 (10%) 10 (17%)  1 (2%) 5 (9%)  0 (0%) 1Cycle 23 7 (30%) 2 (9%)  3 (13%) 0 (0%) 4 (17%) 0 (0%) 2 Cycle 7 3 (43%)2 (29%) 1 (14%)  1 (14%) 3 (43%) 0 (0%) 3-9

Infections occurred in 11 (19%) patients, including 4 serious events[fatal septic entercolitis from pre-study pancreatectomy (Sumpsyndrome); fatal septic bacteremia from unidentified source;post-interventional Grade 3 acute cholangitis; Grade 3 pneumoniaresponding to antibiotics]; and 10 Grade 1-2 events [upper respiratoryinfection (URI)×4, urinary tract infection (UTI)×3, superficial fungalinfection×2, pneumonia, Lyme disease). The fatal bacteremia wasconsidered possibly-related although the patient had a history of severeinfections and recent biliary stent placement; other infections wereconsidered unrelated by the investigators. Furthermore, only 3 patientswere neutropenic (700-900 cells/μL) at time of infection (pneumonia,UTI, URI).

Bleeding occurred in 9 (16%) patients, including 3 serious events (fatalGI bleeding from consumptive coagulopathy, Grade 3 melena fromconcomitant medications, Grade 3 GI bleeding from underlying disease),and 7 minor Grade 1 events (bruising×4, epistaxis, hemorrhoids,conjunctival). Minor bruising was considered at least possibly studydrug related, but the other bleeding events were all consideredunrelated by the investigators, and only 3 patients had Grade 3-4thrombocytopenia at time of event (consumptive coagulopathy,bruising×2).

Efficacy.

The median overall survival (OS) for all 58 patients was 2.7 months,with 2 patients (both Arm A) currently alive 22 and 23 months fromtreatment initiation. Kaplan-Meier survival curves (FIG. 7) showedimprovement in Arm A v B beginning at about 3 months, with the relativenumber of patients remaining alive in Arm A v B then progressivelyincreasing with time (hazard ratio [HR] 0.55, 95% CI: 0.29-0.86;P=0.017, log-rank test). Median OS for Arm A v B (Table 6) was only 2.7v 2.6 months overall, but increased to 7.9 v 3.4 months for thosepatients who received multiple cycles (HR 0.32, P=0.004), including 3patients in Arm A surviving >1 year (one for 1.5 years, the others stillalive). OS generally increased with better performance status and lowerCA19-9 serum levels at study entry, and to a lesser degree with fewerprior therapies and smaller tumor burden estimated by summing lengths ofindex lesions (Table 6).

TABLE 6 Dependence of overall survival (OS) on treatment and patientfactors N Median (range), months Treatment Arm A, Overall 29 2.7(0.4-22.8+) Single Cycle 17 1.9 (0.4-11.0) Multiple Cycles 12 7.9(3.9-22.8+) Arm B, Overall 29 2.6 (0.7-9.4) Single Cycle 18 1.7(0.7-4.1) Multiple Cycles 11 3.4 (1.7-9.4) Patient Factors KarnofskyPerformance Status  90-100 24 4.0 (0.4-22.8+) 70-80 34 2.0 (0.7-21.7+)Number of Prior Systemic Treatments 2 19 2.9 (0.4-21.7+) 3 18 3.1(0.9-22.8+) >3 21 2.4 (0.7-17.5) Sum of Index Lesions (cm) 1.2-8.1 292.9 (0.7-22.8+)  8.3-22.2 29 2.6 (0.4-21.7+) Serum CA19-9 (U/mL) ≦125729 3.9 (0.4-22.8+) >1257 28* 2.1 (0.7-8.4) f.n. +indicates value frompatient currently alive *Baseline CA19-9 unavailable in one patient.

There was >25% disease control (PR+SD) in both treatment arms at interimevaluation, thus meeting the SWOG two-stage criteria to completeenrollment. By RECIST criteria, there were 2 PRs (both Arm A) and 22 SDs(10 Arm A, 12 Arm B) as best response, with other patients havingprogressed by first CT evaluation 4 weeks after cycle 1. Median OS forthose with PR, SD and PD was 11.5, 5.1 and 1.8 months, respectively.Eleven patients had elevated CA19-9 levels at baseline that decreasedwith treatment, either 20-50% and considered a minor response (N=8),or >50% and considered an objective response (N=3). Median OS forpatients with CA19-9 responses was 3.9 v 2.5 months for the remainingpopulation.

Immunogenicity.

Five patients (1 arm A, 4 arm B) with baseline serum samples that wereHAHA-negative (<50 ng/mL) became HAHA-positive after their first (N=3)or second (N=2) cycle, developing maximum titers of 135-21,611 ng/mL.These were isolated laboratory findings without event and of uncertainclinical significance.

Discussion

Despite having 3 median (2 to 7) prior chemotherapies, 58 patients withmetastatic pancreatic cancer were enrolled within 8 months,demonstrating the feasibility of using ⁹⁰Y-clivatuzumab tetraxetan inthis setting. All had received gemcitabine-containing regimens (21% withnab-paclitaxel), and 97% had received fluoropyrimidine-containingregimens (50% with FOLFIRINOX), underscoring the importance ofdeveloping treatments beyond 2nd line for pancreatic cancer.

There were no infusion reactions and, as expected, cytopenias(predominantly thrombocytopenia) were the only significant toxicities.Even in this population, these were mostly transient and reversibleevents, with infrequent hematologic support required. Treatment-relatedmyelosuppression may have exacerbated two cases of consumptivecoagulopathy, but otherwise, the few infections or major bleeding eventsthat occurred could be attributed to complications of underlyingdisease. Most other AEs were mild-moderate constitutional andgastrointestinal events also expected in advanced pancreatic cancer, andcomparison of events between treatment arms showed no substantialdifferences. Thus this combination approach appears to be an acceptableregimen in this advanced population.

Although median survival was only 2.7 months overall, the addition oflow-dose gemcitabine to this regimen showed survival progressivelyimproving with time (48% v 35% alive at 3 months, 35% v 10% at 6 months,21% v 3% at 9 months, 10% v 0% at 1 year). For patients receiving onlyone cycle, gemcitabine made little difference, but with multiple cyclesmedian OS increased (3.4 v 7.9 months), including 3 patientssurviving >1 year. Patients undergoing multiple cycles could reflect ahealthier population, but this would not explain survival resultsfavoring combining radioimmunotherapy with low-dose gemcitabine.Although more limited than previously seen with ⁹⁰Y-clivatuzumabtetraxetan in less heavily treated patients (Gulec et al., 2011, ClinCancer Res 17:4091-100; Ocean et al., 2012, Cancer 118:5497-505), tumorresponse assessed by CT imaging or CA19-9 levels still showed treatmentactivity, and the improvement of survival with better responses andpatient risk factors also supported the consistency of results in thisadvanced population. However, to additionally examine the role oflow-dose gemcitabine in the treatment regimen before pursuing a large,randomized, controlled trial, the study was powered to demonstrateprespecified criteria for treatment arm activity, not survival.

In conclusion, this trial demonstrated the feasibility of using⁹⁰Y-clivatuzumab tetraxetan in metastatic pancreatic cancer patientsafter ≧2 prior chemotherapy regimens (3^(rd) line and beyond). This isimportant, because the benefits of current second-line therapies appearmodest, at the cost of drug toxicity (Rahma et al., 2013, Ann Oncol24:1972-9; Seufferlein et al., 2012, Ann Oncol 23(suppl 7):vii33-40;Almhanna & Kim, 2008, Oncology (Williston Park) 22:1176-83).⁹⁰Y-clivatuzumab tetraxetan combined with low-dose gemcitabine appearspromising in this difficult-to-treat population.

Example 3 Humanized PAM4 MAb

In preferred embodiments, the claimed methods and compositions utilizethe antibody hPAM4 which is a humanized IgG of the murine PAM4 MAbraised against pancreatic cancer mucin. Humanization of the murine PAM4sequences was utilized to reduce the human antimouse antibody (HAMA)response. To produce the humanized PAM4, murine complementaritydetermining regions (CDR) were transferred from heavy and light variablechains of the mouse immunoglobulin into human framework region (FR)antibody sequences, followed by the replacement of some human FRresidues with their murine counterparts. Humanized monoclonal antibodiesare suitable for use in in vitro and in vivo diagnostic and therapeuticmethods.

Comparison of the variable region framework sequences of the murine PAM4MAb (FIG. 8A and FIG. 8B) to known human antibodies in the Kabatdatabase showed that the FRs of PAM4 VK and V_(H) exhibited the highestdegree of sequence homology to that of the human antibodies Walker VK(FIG. 10A) and Wil2 V_(H) (FIG. 10B), respectively. Therefore, theWalker VK (FIG. 10A) and Wil2 V_(H) (FIG. 10B) FRs were selected as thehuman frameworks into which the murine CDRs for PAM4 VK and V_(H) weregrafted, respectively. The FR4 sequence of the human antibody, NEWM,however, was used to replace the Wil2 FR4 sequence for the humanizationof the PAM4 heavy chain (FIG. 10B). A few amino acid residues in PAM4FRs that flank the putative CDRs were maintained in hPAM4 based on theconsideration that these residues have more impact on Ag binding thanother FR residues. These residues were 21M, 47W, 59P, 60A, 85S, 87F, and100G of VK (FIG. 10A) and 27Y, 30P, 38K, 48I, 66K, 67A, and 69L of V_(H)(FIG. 10B). The DNA and amino acid sequences of hPAM4 VK (SEQ ID NO:16)and V_(H) (SEQ ID NO: 19) are shown in FIGS. 11A and 11B, respectively.

A modified strategy as described by Leung et al. (Leung et al., 1994))was used to construct the designed VK (FIG. 11A) and V_(H) (FIG. 11B)genes for hPAM4 using a combination of long oligonucleotide synthesesand PCR. For the construction of the hPAM4 V_(H) domain, two longoligonucleotides, hPAM4 V_(H) A (173-mer) and hPAM4 V_(H) B (173-mer)were synthesized on an automated DNA synthesizer (Applied Biosystems).hPAM4 V_(H) A represents nt 17 to 189 of the hPAM4 V_(H) domain.

(SEQ ID NO: 57) 5′-AGTCTGGGGC TGAGGTGAAG AAGCCTGGGG CCTCAGTGAAGGTCTCCTGC GAGGCTTCTG GATACACATT CCCTAGCTAT GTTTTGCACT GGGTGAAGCAGGCCCCTGGA CAAGGGCTTG AGTGGATTGG ATATATTAAT CCTTACAATG ATGGTACTCAGTACAATGAG AAG-3′

hPAM4 V_(H)B represents the minus strand of the hPAM4 V_(H) domaincomplementary to nt 169 to 341.

(SEQ ID NO: 58) 5′-AGGGTTCCCT GGCCCCAGTA AGCAAATCCG TAGCTACCACCGAAGCCTCT TGCACAGTAA TACACGGCCG TGTCGTCAGA TCTCAGCCTG CTCAGCTCCATGTAGGCTGT GTTGATGGAC GTGTCCCTGG TCAGTGTGGC CTTGCCTTTG AACTTCTCATTGTACTGAGT ACC-3′

The 3′-terminal sequences (21 nt residues) of hPAM4 V_(H)A and V_(H)Bare complementary to each other. Under defined PCR condition, the3′-ends of hPAM4 V_(H)A and V_(H)B anneal to form a short doublestranded DNA flanked by the rest of the long oligonucleotides. Eachannealed end serves as a primer for the transcription of the singlestranded DNA, resulting in a double strand DNA composed of the nt 17 to341 of hPAM4 V_(H). This DNA was further amplified in the presence oftwo short oligonucleotides, hPAM4 V_(H)BACK and hPAM4 V_(H)FOR to formthe full-length hPAM4 V_(H). The underlined portions are restrictionsites for subcloning as shown in FIG. 11B.

hPAM4 V_(H)BACK (SEQ ID NO: 59) 5′-CAG GTG CAG CTG CAG CAG TCT GGG GCTGAG GTG A-3′ hPAM4 V_(H)FOR (SEQ ID NO: 60) 5′-TGA GGA GAC GGT GAC CAGGGT TCC CTG GCC CCA-3′

A minimal amount of hPAM4 V_(H)A and V_(H)B (determined empirically) wasamplified in the presence of 10 μL of 10×PCR Buffer (500 mM KCl, 100 mMTris HCl buffer, pH 8.3, 15 mM MgCl₂), 2 μmol of hPAM4 V_(H)BACK andhPAM4 V_(K)FOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus,Norwalk, Conn.). This reaction mixture was subjected to three cycles ofpolymerase chain reaction (PCR) consisting of denaturation at 94° C. for1 minute, annealing at 45° C. for 1 minute, and polymerization at 72° C.for 1.5 minutes. This procedure was followed by 27 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at55° C. for 1 minute, and polymerization at 72° C. for 1 minute.Double-stranded PCR-amplified product for hPAM4 V_(H) was gel-purified,restriction-digested with PstI and BstEII restriction sites and clonedinto the complementary PstI/BstEII restriction sites of the heavy chainstaging vector, V_(H)pBS2, in which the V_(H) sequence was fullyassembled with the DNA sequence encoding the translation initiationcodon and a secretion signal peptide in-frame ligated at the 5′-end andan intron sequence at the 3′-end. V_(H)pBS2 is a modified staging vectorof V_(H)pBS (Leung et al., Hybridoma, 13:469, 1994), into which a XhoIrestriction site was introduced at sixteen bases upstream of thetranslation initiation codon to facilitate the next subcloning step. Theassembled V_(H) gene was subcloned as a XhoI-BamHI restriction fragmentinto the expression vector, pdHL2, which contains the expressioncassettes for both human IgG heavy and light chains under the control ofIgH enhancer and MT1 promoter, as well as a mouse d/fr gene as a markerfor selection and amplification. Since the heavy chain region of pdHL2lacks a BamHI restriction site, this ligation requires use of a linkerto provide a bridge between the BamHI site of the variable chain and theHindIII site present in the pdHL2 vector. The resulting expressionvectors were designated as hPAM4 V_(H)pdHL2.

For constructing the full length DNA of the humanized VK sequence (FIG.11A), hPAM4 V_(K)A (157-mer) and hPAM4 V_(K)B (156-mer) were synthesizedas described above. hPAM4 V_(K)A and V_(K)B were amplified by two shortoligonucleotides hPAM4 V_(K)BACK and hPAM4 V_(K)FOR as described above.hPAM4 V_(K)A represents nt 16 to 172 of the hPAM4 VK domain.

(SEQ ID NO: 61) 5′-CAGTCTCCAT CCTCCCTGTC TGCATCTGTA GGAGACAGAGTCACCATGAC CTGCAGTGCC AGCTCAAGTG TAAGTTCCAG CTACTTGTAC TGGTACCAACAGAAACCAGG GAAAGCCCCC AAACTCTGGA TTTATAGCAC ATCCAACCTG GCTTCTG-3′

hPAM4 V_(K)B represents the minus strand of the hPAM4 VK domaincomplementary to nt 153 to 308.

(SEQ ID NO: 62) 5′-GTCCCCCCTC CGAACGTGTA CGGGTACCTA TTCCACTGATGGCAGAAATA AGAGGCAGAA TCTTCAGGTT GCAGACTGCT GATGGTGAGA GTGAAGTCTGTCCCAGATCC ACTGCCACTG AAGCGAGCAG GGACTCCAGA AGCCAGGTTG GATGTG-3′

The 3′-terminal sequences (20 nt residues) of hPAM4 V_(K)A and V_(K)Bare complementary to each other. Under defined PCR condition, the3′-ends of hPAM4 V_(K)A and V_(K)B anneal to form a shortdouble-stranded DNA flanked by the rest of the long oligonucleotides.Each annealed end served as a primer for the transcription of the singlestranded DNA, resulting in a double strand DNA composed of nt 16 to 308of hPAM4 VK. This DNA was further amplified in the presence of two shortoligonucleotides, hPAM4 V_(K)BACK and hPAM4 V_(K)FOR to form thefull-length hPAM4 VK. The underlined portions are restriction sites forsubcloning as described below.

hPAM4 V_(K)BACK (SEQ ID NO: 63) 5′-GAC ATC CAG CTG ACC CAG TCT CCA TCCTCC CTG-3′ hPAM4 V_(K)FOR (SEQ ID NO: 64) 5′-TTA GAT CTC CAG TCG TGT CCCCCC TCC GAA CGT-3′

Gel-purified PCR products for hPAM4 VK were restriction-digested withPvuII and BglII and cloned into the complementary PvuII/BclI sites ofthe light chain staging vector, V_(K)pBR2. V_(K)pBR2 is a modifiedstaging vector of V_(K)pBR (Leung et al., Hybridoma, 13:469, 1994), intowhich a XbaI restriction site was introduced at sixteen bases upstreamof the translation initiation codon. The assembled VK genes weresubcloned as XbaI-BamHI restriction fragments into the expression vectorcontaining the V_(H) sequence, hPAM4 V_(H)pdHL2. The resultingexpression vectors were designated as hPAM4pdHL2.

Approximately 30 μg of hPAM4pdHL2 was linearized by digestion with SalIand transfected into Sp2/0-Ag14 cells by electroporation at 450 V and 25μF. The transfected cells were plated into 96-well plates and incubatedin a CO₂ cell culture incubator for two days and then selected for MTXresistance. Colonies surviving selection emerged in two to three weeksand were screened for human antibody secretion by ELISA assay. Briefly,supernatants (˜100 ul) from the surviving colonies were added into thewells of an ELISA microplate precoated with goat anti-human IgG F(ab′)₂fragment-specific Ab. The plate was incubated for one hour at roomtemperature. Unbound proteins were removed by washing three times withwash buffer (PBS containing 0.05% Tween-20). Horseradishperoxidase-conjugated goat anti-human IgG Fc fragment-specific Ab wasadded to the wells. Following incubation for one hour, a substratesolution (100 μL/well) containing 4 mM o-phenylenediaminedihydrochloride (OPD) and 0.04% H₂O₂ in PBS was added to the wells afterwashing. Color was allowed to develop in the dark for 30 minutes and thereaction was stopped by the addition of 50 μL of 4 N H₂SO₄ solution. Thebound human IgG was measured by reading the absorbance at 490 nm on anELISA reader. Positive cell clones were expanded and hPAM4 was purifiedfrom cell culture supernatant by affinity chromatography on a Protein Acolumn.

The Ag-binding activity of hPAM4 was confirmed by ELISA assay in amicrotiter plate coated with pancreas cancer cell extracts. An ELISAcompetitive binding assay using PAM4-antigen coated plates was developedto assess the Ag-binding affinity of hPAM4 in comparison with that of achimeric PAM4 composed of murine V and human C domains. Constant amountsof the HRP-conjugated cPAM4 mixed with varying concentrations of cPAM4or hPAM4 were added to the coated wells and incubated at roomtemperature for 1-2 h. The amount of HRP-conjugated cPAM4 bound to theCaPan1 Ag was revealed by reading the absorbance at 490 nm after theaddition of a substrate solution containing 4 mM o-phenylenediaminedihydrochloride and 0.04% H₂O₂. As shown by the competition assays inFIG. 12, hPAM4 and cPAM4 antibodies exhibited similar bindingactivities.

Example 4 Immunohistochemistry Staining Studies

Immunohistochemistry on normal adult tissues showed that the PAM4reactive epitope was restricted to the gastrointestinal tract wherestaining was weak, yet positive (Table 7). Normal pancreatic tissue,including ducts, ductules, acini, and islet cells, were negative forstaining. A PAM4 based enzyme immunoassay with tissue homogenates asantigens generally supported the immunohistology data (Table 8). ThePAM4 epitope was absent from normal pancreas and othernon-gastrointestinal tissues. In neoplastic tissues, PAM4 was reactivewith twenty one out of twenty five (85%) pancreatic cancers (Table 9 andTable 10) and ten out of twenty six colon cancers, but only limitedreactivity with tumors of the stomach, lung, breast, ovary, prostate,liver or kidney (Table 10). PAM4 reactivity appeared to correlate withthe stage of tumor differentiation, with a greater percentage ofstaining observed in well differentiated pancreatic cancers than inmoderately differentiated or poorly differentiated tumors. Generally,poorly differentiated tumors represent less than 10% of all pancreaticcancers.

These studies have shown the PAM4 reactivity and tissue distribution(both normal and cancer) to be unlike that reported for the CA19.9,DUPAN2, SPAN1, Nd2 and B72.3 antibodies and antibodies against the Lewisantigens. Together with crossblocking studies performed with certain ofthese MAbs, the data suggests that the PAM4 MAb recognizes a unique andnovel epitope. When compared to the antigens recognized by the CA19.9,DUPAN2, and anti-Le^(a) antibodies, the PAM4 antigen appears to be morerestricted in its tissue distribution and is reactive with a higherpercentage of pancreatic tumors. Moreover, it gives a greater overallintensity of reaction at equivalent concentrations and is reactive witha higher percentage of cells within the pancreatic tumors. Finally, PAM4was found to be only weakly reactive with three out of twelve chronicpancreatitis specimens, whereas CA19.9 and DUPAN2 were strongly reactivewith all twelve specimens. Although it is recognized that specificity isdependent in part upon the type of assay employed and the range andnumber of tissues examined, the ability of PAM4 to discriminate betweennormal and neoplastic pancreatic tissue, its ability to react with alarge percentage of the cancer specimens, the high intensity of thereactions, and the ability to distinguish between early stage pancreaticcancer and benign conditions such as pancreatitis are importantcharacteristics of this exemplary anti-pancreatic cancer antibody.

TABLE 7 Immunoperoxidase Staining of Normal Adult Tissues with MAb PAM4Staining Tissue Reaction Pancreas (22)^(a) — Ducts — Acini — Islets —Submaxillary gland (2) — Esophagus (2) — Stomach (3) + mucus secretingcells Duodenum (3) + goblet cells Jejunum (3) + goblet cells Ileum (3) +goblet cells Colon (5) + goblet cells Liver (3) — Gallbladder (2) —Bronchus (3) — Lung (3) — Heart (3) — Spleen (3) — Kidney (3) — Bladder(3) — Prostate (2) — Testes (2) — Uterus (2) — Ovary (2) — ^(a)number ofindividual specimens examined in parentheses

TABLE 8 Monoclonal Antibody PAM4 Reactivity with Normal Adult TissueHomogenates by EIA Tissue μg/g tissue^(a) Pancreas 6.4 Esophagus 8.1Stomach 61.3 Duodenum 44.7 Jejunum 60.6 Colon 74.5 Liver 0.0 Gallbladder5.6 Heart 3.7 Spleen 3.4 Kidney 6.6 Bladder 4.9 Thyroid 3.5 Adrenal 1.3Ureter 2.6 Testes 3.9 CaPan1 Pancreatic Tumor 569 ^(a)values are meanfrom two autopsy specimens

TABLE 9 Immunohistochemical Reactivity of Several Monoclonal Antibodieswith Pancreatic Tumors Differentiation PAM4 CA19.9 Le^(a) DUPAN2 1 W +++− − +++ 2 M ++ +++ +++ + 3 M + − + + 4 M +++ +++ +++ + 5 M ++ + − − 6M + ND ND ND 7 M* +++ +++ +++ +++ 8 M + − − +++ 9 M ++ + ++ − 10 M* ++++ ++ +++ 11 M ++ +++ +++ + 12 M ++ + + +++ 13 M + +++ +++ + 14 M ++ + +++ 15 M +++ + + ++ 16 M + + ++ − 17 M − + + − 18 M ++ ++ ++ ++ 19 M+++ + +++ ++ 20 M + − − − 21 M +++ +++ + ++ 22 P + + + +++ 23 P − − − −24 P − − − − 25 P − − + − TOTAL 21/25 17/24 18/24 16/24 : Negative; +:5-20% of tissue is stained; ++: 21-50% of tissue is stained; +++: >50%of tissue is stained; W, M, P: Well, moderate, or poor differentiation;: Metastatic tissue; ND: Not Done

TABLE 10 Immunoperoxidase Staining of Neoplastic Tissues with MAb PAM4Cancer Tissue Positive/Total Pancreas 21/25 Colon 10/26 Stomach 1/5 Lung 1/15 Breast  0/30 Ovarian  0/10 Prostate 0/4 Liver  0/10 Kidney 0/4

Example 5 In Vivo Biodistribution and Tumor Targeting of RadiolabeledPAM4

Initial biodistribution studies of PAM4 were carried out in a series offour different xenografted human pancreatic tumors covering the range ofexpected differentiation. Each of the four tumor lines employed, AsPc1,BxPc3, Hs766T and CaPan1, exhibited concentrations of ¹³¹I-PAM4 withinthe tumors (range: 21%-48% ID/g on day three) that were significantly(P<0.01-0.001) higher than concomitantly administered nonspecific,isotype-matched Ag8 antibody (range: 3.6%-9.3% ID/g on day three). Thebiodistribution data were used to estimate potential radiation doses tothe tumor of 12,230; 10,684; 6,835; and 15,843 cGy/mCi of injected doseto AsPc1, BxPc3, Hs766T and CaPan1, respectively. With an actual maximumtolerated dose (MTD) of 0.7 mCi, PAM4 could provide substantial rad doseto each of the xenografted tumor models. In each tumor line the bloodlevels of radiolabeled PAM4 were significantly (P<0.01-0.001) lower thanthe nonspecific Ag8. Potential radiation doses to the blood from PAM4were 1.4-4.4 fold lower than from Ag8. When radiation doses to the tumorfrom PAM4 were normalized to the blood doses from PAM4, the tumorsreceived doses that were 2.2; 3.3; 3.4; and 13.1-fold higher than blood,respectively. Importantly, potential radiation doses to non-tumortissues were minimal.

The biodistribution of PAM4 was compared with an anti-CEA antibody,MN-14, using the CaPan1 tumor model. The concentration of PAM4 withinthe tumor was much greater than MN-14 at early timepoints, yieldingtumor:blood ratios at day three of 12.7±2.3 for PAM4 compared to 2.7±1.9for MN-14. Although PAM4 uptake within the tumor was significantlyhigher than for MN-14 at early timepoints (day one—P<0.001; daythree—P<0.01), dosimetry analyses indicated only a 3.2-fold higher doseto the tumor from PAM4 as compared to MN-14 over the fourteen day studyperiod. This was due to a rapid clearance of PAM4 from the tumor, suchthat at later timepoints similar concentrations of the two antibodieswere present within the tumors. A rapid clearance of PAM4 from the tumorwas also noted in the BxPc3 and Hs766T but not AsPc1 tumor models. Theseobservations were unlike those reported for other anti-mucin antibodies,as for example G9 and B72.3 in colorectal cancer, where each exhibitedlonger retention times as compared to the MN-14 antibody. Results fromstudies on the metabolism of PAM4, indicate that after initial bindingto the tumor cell, antibody is rapidly released, possibly beingcatabolized or being shed as an antigen:antibody complex. The bloodclearance is also very rapid. These data suggest that ¹³¹I may not bethe appropriate choice of isotope for therapeutic applications. Ashort-lived isotope, such as ⁹⁰Y or ¹⁸⁸Re, which can be administeredfrequently may be a more effective reagent.

PAM4 showed no evidence of targeting to normal tissues, except in theCaPan1 tumor model, where a small but statistically significant splenicuptake was observed (range 3.1-7.5% ID/g on day-3). This type of splenictargeting has been observed in the clinical application of theanti-mucin antibodies B72.3 and CC49. Importantly, these studies alsoreported that splenic targeting did not affect tumor uptake of antibodynor did it interfere with interpretation of the nuclear scans. Thesestudies suggested that splenic targeting was not due to crossreactiveantigens in the spleen, nor to binding by Fc receptors, but rather toone or more of the following possibilities: direct targeting of antigentrapped in the spleen, or indirect uptake of antigen:antibody complexesformed either in the blood or released from the tumor site. The latterwould require the presence of immune complexes in the blood. However,these were not observed when specimens as early as five minutes and aslate as seven days were examined by gel filtration (HPLC, GF-250column); radiolabeled antibody eluted as native material. The formerexplanation seems more likely in view of the fact that the CaPan1 tumorproduced large quantities of PAM4-reactive antigen, 100- to 1000-foldhigher than for the other tumor cell lines examined. The lack of splenictargeting by PAM4 in these other tumor lines suggests that thisphenomenon was related to excessive antigen production. Splenictargeting can be overcome by increasing the protein dose to 10 μg fromthe original 2 μg dose. A greater amount of the splenic entrappedantigen presumably was complexed with unlabeled PAM4 rather thanradiolabeled antibody. Increasing the protein dose had no adverse effectupon targeting of PAM4 to the tumor or nontumor tissues. In fact, anincrease of the protein dose to 100 μg more than doubled theconcentration of radiolabeled PAM4 within the CaPan1 tumor.

Example 6 Development of Orthotopic Pancreatic Tumor Model in AthymicNude Mice

In order to resemble the clinical presentation of pancreatic cancer inan animal model more closely, we developed an orthotopic model byinjecting tumor cells directly into the head of the pancreas. OrthotopicCaPan1 tumors grew progressively without overt symptoms until thedevelopment of ascites and death at ten to fourteen weeks. By three tofour weeks post-implantation, animals developed a palpable tumor ofapproximately 0.2 g. Within eight weeks of growth, primary tumors ofapproximately 1.2 g along with metastases to the liver and spleen wereobserved (1-3 metastatic tumors/animal; each tumor <0.1 g). At ten tofourteen weeks seeding of the diaphragm with development of ascites wereevident. Ascites formation and occasional jaundice were usually thefirst overt indications of tumor growth. At this time tumors were quitelarge, 1 to 2 g, and animals had at most only three to four weeks untildeath occurred.

Radiolabeled ¹³¹I-PAM4, administered to animals bearing four week oldorthotopic tumors (approximately 0.2 g) showed specific targeting to theprimary tumor with localization indices of 7.9±3.0 at day one increasingto 22.8±15.3 at day fourteen. No evidence of specific targeting to othertissues was noted. In one case where tumor metastases to the liver andspleen were observed, both metastases were targeted, and had highconcentrations of radiolabeled antibody. In addition, approximately halfof the animals developed a subcutaneous tumor at the incision site. Nosignificant differences were noted in the targeting of orthotopic andsubcutaneous tumors within the same animal, and no significantdifferences were observed in the targeting of orthotopic tumor whetheror not the animal had an additional subcutaneous tumor. The estimatedradiation doses from PAM4 were 6,704 and 1,655 cGy/mCi to the primarytumor and blood, respectively.

Example 7 Radioimmunotherapy of Pancreatic Cancer

The initial studies on the use of ¹³¹I-PAM4 for therapy were carried outwith the CaPan1 tumor, which was grown as a subcutaneous xenograft inathymic mice. Animals bearing a 0.25 g tumor were administered 350 μCi,¹³¹I-PAM4 in an experiment that also compared the therapeutic effects ofa similar dose of nonspecific Ag8. The MTD for administration of¹³¹I-PAM4 to animals bearing 1 cm³ tumors is 700 μCi. By weeks five andsix, the PAM4 treated animals showed a dramatic regression of tumor, andeven at week twenty seven, five out of eight remained tumor free. Theuntreated, as well as Ag8-treated animals, showed rapid progression oftumor growth although a significant difference was noted between thesetwo control groups. At seven weeks, tumors from the untreated group hadgrown 20.0±14.6-fold from the initial timepoint whereas the¹³¹I-Ag8-treated tumors had grown only 4.9±1.8-fold. At this time point,the PAM4 tumors had regressed to 0.1±0.1-fold of their original size, asignificant difference from both untreated (P<0.001) and nonspecificAg8-treated (P<0.01) animals.

These data show that CaPan1 tumors were sensitive to treatment with¹³¹I-PAM4. The outcome, that is, regression or progression of the tumor,was dependent upon several factors including initial tumor size. Thus,groups of animals bearing CaPan1 tumor burdens of 0.25 g, 0.5 g, 1.0 g,or 2.0 g were treated with a single dose of the 350 μCi ¹³¹I-PAM4. Themajority of animals having tumors of initial size 0.25 g and 0.5 g (nineof ten animals in each group) showed tumor regression or growthinhibition for at least sixteen weeks post treatment. In the 1.0 g tumorgroup five out of seven showed no tumor growth for the sixteen weekperiod and in the 2.0 g tumor group six out of nine showed no tumorgrowth for a period of six weeks before progression occurred. Although asingle 350 μCi dose was not as effective against larger tumors, a singledose may not be the appropriate regimen for large tumors.

Toxicity studies indicate the ability to give multiple cycles ofradioimmunotherapy, which may be more effective with a larger tumorburden. Animals bearing CaPan1 tumors averaging 1.0 g, were given eithera single dose of 350 μCi ¹³¹I-PAM4, two doses given at times zero andfour weeks or were left untreated. The untreated group had a meansurvival time of 3.7±1.0 weeks (survival defined as time for tumor toreach 5 cm³). Animals died as early as three weeks, with no animalsurviving past six weeks. A single dose of 350 μCi ¹³¹I-PAM4 produced asignificant increase in the survival time to 18.8±4.2 weeks (P<0.0001).The range of animal deaths extended from weeks thirteen to twenty five.None of the animals were alive at the end of the study period of twentysix weeks.

A significant increase in survival time was observed for the two dosegroup as compared to the single dose group. Half of the animals werealive at the twenty six week timepoint with tumor sizes from 1.0-2.8cm³, and a mean tumor growth rate of 1.6±0.7 fold from initial tumorsize. For those animals that were non-survivors at twenty six weeks, themean survival time (17.7±5.3 weeks) was similar to the single dosegroup.

Therapy studies with PAM4 were also conducted using the orthotopic tumormodel. Groups of animals bearing four week old orthotopic tumors(estimated tumor weight of 0.25 g) were either left untreated or treatedwith a single dose of either 350 μCi ¹³¹I-PAM4 or 350 μCi of¹³¹I-nonspecific Ag8. The untreated animals had a 50% death rate by weekten with no survivors at week fifteen. Animals administered nonspecific¹³¹I-Ag8 at four weeks of tumor growth, showed a 50% death rate at weekseven with no survivors at week fourteen. Although statistically(logrank analysis) there were no differences between these two groups,it is possible that radiation toxicity had occurred in the Ag8 treatedanimals. Radiolabeled PAM4 provided a significant survival advantage(P<0.001) as compared to the untreated or Ag8 treated animals, with 70%survival at sixteen weeks, the end of the experiment. At this time thesurviving animals were sacrificed to determine tumor size. All animalshad tumor with an average weight of 1.2 g, as well as one or two small(<0.1 g) metastases evident in four of the seven animals. At sixteenweeks of growth, these tumors were more representative of aneight-week-old tumor.

Example 8 Combined Modality GEMZAR® Chemotherapy and ¹³¹I-PAM4Experimental Radioimmunotherapy

Initial studies into the combined use of gemcitabine (GEMZAR®) with¹³¹I-PAM4 radioimmunotherapy were performed as a checkerboard array; asingle dose of gemcitabine (0, 100, 200, 500 mg/kg) versus a single doseof ¹³¹I-PAM4 ([MTD=700 μCi] 100%, 75%, 50%, 0% of the MTD). The combinedMTD was found to be 500 mg/kg gemcitabine with 350 μCi ¹³¹I-PAM4 (50%MTD). Toxicity, as measured by loss of body weight, went to the maximumconsidered as nontoxic; that is 20% loss in body weight. Although thecombined treatment protocol was significantly more effective thangemcitabine alone, the treatment was no more effective thanradioimmunotherapy alone. The next studies were performed at a low doseof gemcitabine and radioimmunotherapy to examine if a true synergistictherapeutic effect would be observed. Athymic nude mice bearing tumorsof approximately 1 cm³ (approximately 5% of body weight) wereadministered gemcitabine, 100 mg/kg on days zero, three, six, nine, andtwelve, with 100 μCi of ¹³¹I-PAM4 given on day zero. A therapeuticeffect was observed with statistically significant (P<0.0001) regression(two of five tumors less than 0.1 cm³) and/or growth inhibition of thetumors compared to gemcitabine alone. Thus, at lower dosages oftherapeutic agent, there surprisingly appears to be a synergistic effectof the combination of gemcitabine and radioimmunotherapy. Of additionalnote, in terms of body weight, toxicity was not observed. Thecombination treatment protocol can, if necessary, be delivered inmultiple cycles, with the second treatment cycle beginning in week-four,as was done with the radioimmunotherapy-alone studies described above.

Example 9 Effects of Reagent Treatment on Immunoreactivity of PAM4Antigen

Treatment of pancreatic mucin with DTT (15 min at room temp), completelyabolished reactivity with PAM4 (DTT-EC₅₀, 0.60±0.0 μM). The onlycysteines (cystine bridges) within MUC-1 are present within thetransmembrane domain and should not be accessible to DTT. The secretedform of MUC-1 does not contain the transmembrane domain and thereforehas no intramolecular cystine bridges. Data from periodate oxidationtreatment of pancreatic cancer mucin with 0.05 M sodium periodate for 2hrs at room temperature yielded 40% loss of immunoreactivity with PAM4antibody (not shown). Further periodate studies have shown as high as a60% loss of immunoreactivity with PAM4 antibody (not shown). The resultsof periodate and DTT studies suggest that the PAM4 epitope isconformationally dependent upon some minimal form of glycosylation, andmay be affected by intermolecular disulfide bond formation.

Example 10 Distribution and Cross-Reactivity of the PAM4 Antigen

The expression of the PAM4-epitope within PanINs is atypical for MUC-1.It is similar to the expression reported for MUC5AC as detected by thecommercially available MAb-CLH2-2. However, an attempted sandwichimmunoassay with PAM4 capture and MAb-CLH2-2 as probe gave negativeresults. Although this possibly suggests the PAM4 and CLH2-2 epitopesmay overlap and thus block each other, the CLH2-2 was reported to bereactive with 42/66 (64%) gastric carcinomas whereas the PAM4 MAb showedreactivity with only 6/40 (15%) of gastric carcinomas and, of these,only in focal reactivity.

Use of the commercially available 45M1, an anti-MUC5AC MAb, as a probereagent in EIA (with PAM4 as capture) provided positive results,indicating that the two epitopes may be present on the same antigenicmolecule. Blocking studies (either direction) indicated that theepitopes bound by 45M1 and PAM4 are in fact two distinct epitopes, as noblocking was observed. Labeling of tissue microarrays consisting ofcores from invasive pancreatic carcinoma has demonstrated significantdifferences for expression of the 45M1 and PAM4 epitopes in individualpatient specimens. Of 28 specimens, concordance was observed in only 17cases (61%). PAM4 was reactive with 24/28 cases (86%) while 45M1 wasreactive with 13/28 (46%) cases (not shown).

The results of periodate studies are consistent with glycosylation as afactor in MUC5AC immunoreactivity with the PAM4 antibody. Thus, resultsof studies with apomucins may not be definitive for antigendetermination.

Although based on EIA capture, the PAM4 antibody appears to bind to thesame antigenic protein as the 45M1 anti-MUC5AC MAb, it is noted thatMUC5AC is not specific to pancreas cancer and it is found in a number ofnormal tissues (other than the gastric mucosa with which PAM4 isreactive). For example, MUC5AC is found in normal lung, colon and othertissues. PAM4 antibody does not bind to normal lung tissues, except asindicated above in few samples and to a limited or minimal amount.

With respect to the effects of DTT and periodate, it is probable thatthe peptide core disulfide bridges are identical no matter what tissueproduces the protein. A specific amino acid sequence should fold in aspecific manner, independent of the tissue source. However,glycosylation patterns may differ dependent upon tissue source.

Example 11 Phage Display Peptide Binding of PAM4 Antibody

PAM4 antibody binding was examined with two different phage displaypeptide libraries. The first was a linear peptide library consisting of12 amino acid sequences and the second was a cyclic peptide consistingof 7 amino acid sequences cyclized by a disulfide bridge. We panned theindividual libraries alternately against hPAM4 and hLL2 (negativeselection with anti-CD22 antibody) for a combined total of 4 rounds, andthen screened the phage displayed peptide for reactivity with both hPAM4and mPAM4 with little to no reactivity against hLL2. Phage binding in anon-specific manner (i.e., binding to epratuzumab [hLL2]) werediscarded.

For the linear phage-displayed peptide, the sequence WTWNITKAYPLP (SEQID NO:7) was identified 30 times (in 35 sequenced phage), each of whichwere shown to have reactivity with PAM4 antibodies. A mutationalanalysis was conducted in which a library based on this sequence andhaving 7.5% degeneracy at each position, was constructed, panned andscreened as before. Variability was noted in the 19 obtained peptidesequences that were positive for PAM4 binding with 7 being identical tothe parental sequence, 5 having the sequence WTWNITKEYPQP (SEQ ID NO:65)and the rest being uniquely present. Table 11 shows the results of thismutational analysis. The upper row lists the sequences identified andthe lower row lists the frequency with which each of the amino acids wasidentified in that position. The parent sequence is most frequent (bold)with the next highest variation a substitution of E for A at position 8and a substitution of Q for L at position 11. It does not appear thatthese substitutions had any great effect upon immunoreactivity.

TABLE 11 Phage Display Amino Acid Sequence (SEQ ID NO: 116) Variationwith Linear Peptide Binding to PAM4 Antibody A K E L R T Q T R I N T N FY P M W T W D I R G C T R C P number of 19 19 19 18 19 17 14 10 18 17 1119 occurrences 1 2 1 5 1 2 5 (out of 19 2 1 1 sequences 1 1 1 analyzed)1 1 1 1

Results with the phage displayed cyclic library were significantlydifferent from the linear library (Table 12). The sequence ACPEWWGTTC(SEQ ID NO:66) was present in 33 of 35 peptide sequences examined.Analysis of the cyclic library presented the following results(positions with an asterisk were invariant and not subject to selectivepressure in the library).

TABLE 12 Phage Display Amino Acid Sequence (SEQ ID NO: 117) Variationwith Linear Peptide Binding to PAM4 Antibody T M T S P G G Q A C Y E W WS S P C number of * * 33 35 35 35 34 29 28 * occurrences 2 1 5 4 (out of19 1 1 sequences 1 analyzed) 1

The two cysteines (at positions 2 and 10) formed a disulfide bridge.Substitution of T at position 9 with any amino acid greatly affectedimmunoreactivity. The sequence GTTGTTC (SEQ ID NO:67) is present withinthe MUC5AC protein towards the amino terminus as compared to the cyclicpeptide sequence shown above, which shows homology at the C-terminal endof the consensus peptide sequence. However, the cyclic peptide onlyshowed approximately 10% of the immunoreactivity of the linear sequencewith the PAM4 antibody. Both linear and cyclic consensus sequences areassociated with a cysteine, which may or may not relate to the effect ofDTT on MUC5AC immunoreactivity.

The results reported herein indicate that the PAM4 epitope is dependentupon a specific conformation which may be produced by disulfide bridges,as well as a specific glycosylation pattern.

Example 12 Immunohistology of Pancreatic Cancer in a PancreatitisSpecimen

Several pathologic conditions predispose patients to the development ofpancreatic carcinoma, such as pancreatitis, diabetes, smoking andothers. Within this pre-selected group of patients, screening measuresare particularly important for the early detection of pancreaticneoplasia. We examined 9 specimens of chronic pancreatitis tissue frompatients having primary diagnosis of this disease. We employed ananti-CD74 MAb, LL1, as an indicator of inflammatory infiltrate, andMAb-MA5 as a positive control for pancreatic ductal and acinar cells.Whereas the two control MAbs provided immunohistologic evidenceconsistent with pancreatitis, in no instance did PAM4 react withinflamed pancreatic tissue. However, in one case, a moderatelydifferentiated pancreatic adenocarcinoma was also present within thetissue specimen. PAM4 gave an intense stain of the neoplastic cellswithin this tumor. In a second case, while the inflamed tissue wasnegative with PAM4, a small PanIN precursor lesion was identified thatwas labeled with PAM4. Labeling of the PanIN within this latter specimenis consistent with early detection of pancreatic neoplasia in a patientdiagnosed with a non-malignant disease. These results show thatdetection and/or diagnosis using the PAM4 antibody may be performed withhigh sensitivity and selectivity for pancreatic neoplasia against abackground of benign pancreatic tissues.

Example 13 Therapy of a Patient with Inoperable and MetastaticPancreatic Carcinoma

Patient 118-001, CWG, is a 63-year-old man with Stage-IV pancreaticadenocarcinoma with multiple liver metastases, diagnosed in November of2007. He agreed to undertake combined radioimmunotherapy and gemcitabinechemotherapy as a first treatment strategy, and was then given a firsttherapy cycle of 6.5 mCi/m² of ⁹⁰Y-hPAM4, combined with 200 mg/m²gemcitabine, whereby the gemcitabine was given once weekly on weeks 1-4and ⁹⁰Y-hPAM4 was given once-weekly on weeks 2-4 (3 doses). Two monthslater, the same therapy cycle was repeated, because no major toxicitieswere noted after the first cycle. Already 4 weeks after the firsttherapy cycle, CT evidence of a reduction in the diameters of theprimary tumor and 2 of the 3 liver metastases surprisingly was noted,and this was consistent with significant decreases in the SUV values ofFDG-PET scans, with 3 of the 4 tumors returning to normal background SUVlevels at this time (FIG. 13 and FIG. 14). The patient's pre-therapyCA-19.9 level of 1,297 dropped to a low level of 77, further supportiveof the therapy being effective. Table 13 shows the effects of combinedradioimmunotherapy with ⁹⁰Y-hPAM4 and gemcitabine chemotherapy in thispatient. It was surprising and unexpected that such low doses of theradionuclide conjugated to the antibody combined with such low,nontoxic, doses of gemcitabine showed such antitumor activity even afteronly a single course of this therapy.

TABLE 13 Effects of Combined Radioimmunotherapy with ⁹⁰Y-hPAM4 andGemcitabine Chemotherapy in Metastatic Pancreatic Carcinoma BaselineLongest 4 wk post-Tx Baseline Diameter Longest PET 4 wk post-Tx TumorLocation (cm) Diameter (cm) (SUV) PET (SUV) Pancreatic tail 4.5 4.3 9.24.2 (primary) L hepatic met 1.9 1.9 4.1 Background R post hepatic 1.71.6 3.7 Background met R central hepatic 1.9 1.2 3.2 Background met

Example 14 Therapy of a Patient with Inoperable Metastatic PancreaticCarcinoma

A 56-year-old male with extensive, inoperable adenocarcinoma of thepancreas, with several liver metastases ranging from 1 to 4 cm indiameter, substantial weight loss (30 lbs of weight or more), mildjaundice, lethargy and weakness, as well as abdominal pains requiringmedication, is given 4 weekly infusions of gemcitabine at doses of 200mg/m² each. On the last three gemcitabine infusions, ⁹⁰Y-DOTA-hPAM4radiolabeled humanized antibody is administered at a dose of 10 mCi/m²of ⁹⁰Y and 20 mg antibody protein, in a two-hour i.v. infusion. Twoweeks later, the patient is given a course of gemcitabine chemotherapyconsisting of 3 weekly doses of 600 mg/m² by i.v. infusion. The patientis then evaluated 4 weeks later, and has a mild leukopenia (grade-2), noother major blood or enzyme changes over baseline, but shows animprovement in the blood CA19.9 titer from 5,700 to 1,200 and a decreasein jaundice, with an overall subjective improvement. This follows 3weeks later with a repeat of the cycle of lower-dose gemcitabine(weekly×4), with 3 doses of ⁹⁰Y-DOTA-hPAM4. Four weeks later, thepatient is reevaluated, and the CT and PET scans confirm anapproximately 40% reduction of total tumor mass (primary cancer andmetastases), with a further reduction of the CA19.9 titer to 870. Thepatient regains appetite and activity, and is able to return to moreusual daily activities without the need for pain medication. He gains 12lbs after beginning this experimental therapy. A repeat of the scans andblood values indicates that this response is maintained 6 weeks later.

Example 15 Preparation of DNL™ Constructs for Pretargeting

DDD and AD Fusion Proteins

The DNL™ technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibodies or fragments thereofor other effector moieties. For certain preferred embodiments, IgGantibodies or Fab antibody fragments may be produced as fusion proteinscontaining either a dimerization and docking domain (DDD) or anchoringdomain (AD) sequence. Although in preferred embodiments the DDD and ADmoieties are produced as fusion proteins, the skilled artisan willrealize that other methods of conjugation, such as chemicalcross-linking or click chemistry, may be utilized within the scope ofthe claimed methods and compositions.

Bispecific antibodies may be formed by combining a Fab-DDD fusionprotein of a first antibody with a Fab-AD fusion protein of a secondantibody. Alternatively, constructs may be made that combine IgG-ADfusion proteins with Fab-DDD fusion proteins. The technique is notlimiting and any protein or peptide of use may be produced as an AD orDDD fusion protein for incorporation into a DNL™ construct. Wherechemical cross-linking is utilized, the AD and DDD conjugates are notlimited to proteins or peptides and may comprise any molecule that maybe cross-linked to an AD or DDD sequence using any cross-linkingtechnique known in the art. In certain exemplary embodiments, apolyethylene glycol (PEG) or other polymeric moiety may be incorporatedinto a DNL™ construct, as described in further detail below.

For pretargeting applications, an antibody or fragment containing abinding site for an antigen associated with a diseased tissue, such as atumor-associated antigen (TAA), may be combined with a second antibodyor fragment that binds a hapten on a targetable construct, to which atherapeutic and/or diagnostic agent is attached. The DNL™-basedbispecific antibody is administered to a subject, circulating antibodyis allowed to clear from the blood and localize to target tissue, andthe conjugated targetable construct is added and binds to the localizedantibody for diagnosis or therapy.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD-fusion protein module can be combined with any AD-fusion proteinmodule to generate a bispecific DNL™ construct. For different types ofconstructs, different AD or DDD sequences may be utilized. Exemplary DDDand AD sequences are provided below.

DDD1: (SEQ ID NO: 68) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2:(SEQ ID NO: 69) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1: (SEQID NO: 70) QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 71) CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 comprise the DDDsequence of the human RIIα form of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 72) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 73) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 74) CGFEELAWKIAKMIWSDVFQQGC

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors. To generate Fab-DDD expression vectors, the codingsequences for the hinge, CH2 and CH3 domains of the heavy chain arereplaced with a sequence encoding the first 4 residues of the hinge, a14 residue Gly-Ser linker and the first 44 residues of human RIIα(referred to as DDD1). To generate Fab-AD expression vectors, thesequences for the hinge, CH2 and CH3 domains of IgG are replaced with asequence encoding the first 4 residues of the hinge, a 15 residueGly-Ser linker and a 17 residue synthetic AD called AKAP-IS (referred toas AD1), which was generated using bioinformatics and peptide arraytechnology and shown to bind RIIα dimers with a very high affinity (0.4nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC (SEQ ID NO: 118)) followedby four glycines and a serine, with the final two codons (GS) comprisinga Bam HI restriction site. The 410 bp PCR amplimer was cloned into thePGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide, designated (G₄S)₂DDD1 (‘(G₄S)₂’ disclosed asSEQ ID NO: 119), was synthesized by Sigma GENOSYS® (Haverhill, UK) tocode for the amino acid sequence of DDD1 preceded by 11 residues of thelinker peptide, with the first two codons comprising a BamHI restrictionsite. A stop codon and an EagI restriction site are appended to the3′end. The encoded polypeptide sequence is shown below.

(SEQ ID NO: 75) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 76) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL™ construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2appended to the carboxyl terminal end of the CH1 domain via a 14 aminoacid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 16 Production of AD- and DDD-Linked Fab and IgG Fusion Proteinsfrom Multiple Antibodies

Using the techniques described in the preceding Example, the IgG and Fabfusion proteins shown in Table 14 were constructed and incorporated intoDNL™ constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL™ constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 14 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 17 Sequence Variants for DNL™

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the DNL™ construct comprise the amino acid sequences of AD1, AD2,AD3, DDD1, DDD2, DDD3 or DDD3C as discussed above. However, inalternative embodiments sequence variants of AD and/or DDD moieties maybe utilized in construction of the DNL™ complexes. For example, thereare only four variants of human PKA DDD sequences, corresponding to theDDD moieties of PKA RIα, RIIα, RIβ and RIIβ. The RIIα DDD sequence isthe basis of DDD1 and DDD2 disclosed above. The four human PKA DDDsequences are shown below. The DDD sequence represents residues 1-44 ofRIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66 of RIβ. (Note that thesequence of DDD1 is modified slightly from the human PKA RIIα DDDmoiety.)

PKA RIα (SEQ ID NO: 77)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ (SEQ ID NO:78) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILA PKA RIIα(SEQ ID NO: 79) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 80) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:68below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

(SEQ ID NO: 68) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:70), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:70. The skilledartisan will realize that in designing sequence variants of the ADsequence, one would desirably avoid changing any of the underlinedresidues, while conservative amino acid substitutions might be made forresidues that are less critical for DDD binding.

AKAP-IS sequence (SEQ ID NO: 70) QIEYLAKQIVDNAIQQA

Gold (2006) utilized crystallography and peptide screening to develop aSuperAKAP-IS sequence (SEQ ID NO:81), exhibiting a five order ofmagnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, which increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL™ constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:82-84. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:68,the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 81) QIEYVAKQIVDYAIHQA Alternative AKAPsequences (SEQ ID NO: 82) QIEYKAKQIVDHAIHQA (SEQ ID NO: 83)QIEYHAKQIVDHAIHQA (SEQ ID NO: 84) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-SPECIFIC AKAPS AKAP-KL (SEQ ID NO: 85) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 86) LLIETASSLVKNAIQLSI AKAP-LBC (SEQ ID NO: 87)LIEEAASRIVDAVIEQVK RI-SPECIFIC AKAPS AKAPCE (SEQ ID NO: 88)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 89) LEQVANQLADQIIKEAT PV38 (SEQ IDNO: 90) FEELAWKIAKMIWSDVF DUAL-SPECIFICITY AKAPS AKAP7 (SEQ ID NO: 91)ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 92) TAEEVSARIVQVVTAEAV DAKAP1 (SEQID NO: 93) QIKQAAFQLISQVILEAT DAKAP2 (SEQ ID NO: 94) LAWKIAKMIVSDVMQQ

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:95-97. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:95), RIAD (SEQ ID NO:96) and PV-38 (SEQ IDNO:97). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 (SEQ ID NO: 95) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 96)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 97) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides are provided in Table 1 of Hundsrucker et al., reproduced inTable 15 below. AKAPIS represents a synthetic RII subunit-bindingpeptide. All other peptides are derived from the RII-binding domains ofthe indicated AKAPs.

TABLE 15 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 70) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO:98) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 99) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 100) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 101) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 102) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 103) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 104) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 105) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 106) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 107) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 108) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 109) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 110) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 111) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 112) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 113) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 114) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 115)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:70). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 70) QIEYLAKQIVDNAIQQA

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:68. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. The skilled artisan will realize that indesigning sequence variants of DDD, it would be most preferred to avoidchanging the most conserved residues (italicized), and it would bepreferred to also avoid changing the conserved residues (underlined),while conservative amino acid substitutions may be considered forresidues that are neither underlined nor italicized.

(SEQ ID NO: 68) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

The skilled artisan will realize that these and other amino acidsubstitutions in the antibody moiety or linker portions of the DNL™constructs may be utilized to enhance the therapeutic and/orpharmacokinetic properties of the resulting DNL™ constructs.

Example 18 Generation of TF2 DNL™ Pretargeting Construct

A trimeric DNL™ construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). The additional bands suggest that disulfideformation is incomplete under the experimental conditions (not shown).Reducing SDS-PAGE shows that any additional bands apparent in thenon-reducing gel are product-related (not shown), as only bandsrepresenting the constituent polypeptides of TF2 were evident (notshown). However, the relative mobilities of each of the fourpolypeptides were too close to be resolved. MALDI-TOF mass spectrometry(not shown) revealed a single peak of 156,434 Da, which is within 99.5%of the calculated mass (157,319 Da) of TF2.

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 g/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of W12 (not shown).

Example 19 Production of TF10 Bispecific Antibody for Pretargeting

A similar protocol was used to generate a trimeric TF10 DNL™ construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The cancer-targeting antibody component in TF10 wasderived from hPAM4, a humanized anti-MUC5AC MAb that has been studied indetail as a radiolabeled MAb (e.g., Gold et al., Clin. Cancer Res. 13:7380-7387, 2007). The hapten-binding component was derived from h679, ahumanized anti-histaminyl-succinyl-glycine (HSG) MAb. The TF10bispecific ([hPAM4]₂×h679) antibody was produced using the methoddisclosed for production of the (anti CEA)₂×anti HSG bsAb TF2, asdescribed above. The TF10 construct bears two humanized PAM4 Fabs andone humanized 679 Fab.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL™ reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

A full tissue histology and blood cell binding panel has been examinedfor hPAM4 IgG and for an anti-CEA×anti-HSG bsMAb that is enteringclinical trials. hPAM4 binding was restricted to very weak binding tothe urinary bladder and stomach in ⅓ specimens (no binding was seen invivo), and no binding to normal tissues was attributed to theanti-CEA×anti-HSG bsMAb. Furthermore, in vitro studies against celllines bearing the H1 and H2 histamine receptors showed no antagonisticor agonistic activity with the IMP 288 di-HSG peptide, and animalstudies in 2 different species showed no pharmacologic activity of thepeptide related to the histamine component at doses 20,000 times higherthan that used for imaging. Thus, the HSG-histamine derivative does nothave pharmacologic activity.

Example 20 Imaging Studies Using Pretargeting with TF10 BispecificAntibody and ¹¹¹In-Labeled Peptides

The following study demonstrates the feasibility of in vivo imagingusing the pretargeting technique with bispecific antibodiesincorporating hPAM4 and labeled peptides. The TF10 bispecific antibody,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679, was prepared as described in the preceding Example. Nudemice bearing 0.2 to 0.3 g human pancreatic cancer xenografts wereimaged, using pretargeting with TF10 and ¹¹¹In-IMP-288 peptide. Theresults, shown in FIG. 15A and FIG. 15B, demonstrate how clearlydelineated tumors can be detected in animal models using a bsMAbpretargeting method, with ¹¹¹In-labeled di-HSG peptide, IMP-288. The sixanimals in the top of FIG. 15A and FIG. 15B received 2 different dosesof TF10 (10:1 and 20:1 mole ratio to the moles of peptide given), andthe next day they were given an ¹¹¹In-labeled diHSG peptide (IMP 288).The 3 other animals on the bottom of FIG. 15A and FIG. 15B received onlythe ¹¹¹In-IMP-288 (no pretargeting). The images shown in FIG. 15B weretaken 3 h after the injection of the labeled peptide and show clearlocalization of 0.2-0.3 g tumors in the pretargeted animals, with nolocalization in the animals given the ¹¹¹In-peptide alone. Tumor uptakeaveraged 20-25% ID/g with tumor/blood ratios exceeding 2000:1,tumor/liver ratios of 170:1, and tumor/kidney ratios of 18/1.

Example 21 Production of Targeting Peptides for Use in Pretargeting and¹⁸F Labeling

In a variety of embodiments, ¹⁸F-labeled proteins or peptides areprepared by a novel technique and used for diagnostic and/or imagingstudies, such as PET imaging. The novel technique for ¹⁸F labelinginvolves preparation of an ¹⁸F-metal complex, preferably an ¹⁸F-aluminumcomplex, which is chelated to a chelating moiety, such as DOTA, NOTA orNETA or derivatives thereof. Chelating moieties may be attached toproteins, peptides or any other molecule using conjugation techniqueswell known in the art. In certain preferred embodiments, the ¹⁸F—Alcomplex is formed in solution first and then attached to a chelatingmoiety that is already conjugated to a protein or peptide. However, inalternative embodiments the aluminum may be first attached to thechelating moiety and the ¹⁸F added later.

Peptide Synthesis

Peptides were synthesized by solid phase peptide synthesis using theFmoc strategy. Groups were added to the side chains of diamino aminoacids by using Fmoc/Aloc protecting groups to allow differentialdeprotection. The Aloc groups were removed by the method of Dangles et.al. (J. Org. Chem. 1987, 52:4984-4993) except that piperidine was addedin a 1:1 ratio to the acetic acid used. The unsymmetrical tetra-t-butylDTPA was made as described in McBride et al. (US Patent Application Pub.No. 2005/0002945, the Examples section of which is incorporated hereinby reference).

The tri-t-butyl DOTA, symmetrical tetra-t-butyl DTPA, ITC-benzyl DTPA,p-SCN-Bn-NOTA and TACN were obtained from MACROCYCLICS® (Dallas, Tex.).The DiBocTACN, NODA-GA(tBu)₃ and the NO2AtBu were purchased fromCheMatech (Dijon, France). The Aloc/Fmoc Lysine and Dap(diaminopropionic acid derivatives (also Dpr)) were obtained fromCREOSALUS® (Louisville, Ky.) or BACHEM® (Torrance, Calif.). The SieberAmide resin was obtained from NOVABIOCHEM® (San Diego, Calif.). Theremaining Fmoc amino acids were obtained from CREOSALUS®, BACHEM®,PEPTECH® (Burlington, Mass.), EMD BIOSCIENCES® (San Diego, Calif.), CHEMIMPEX® (Wood Dale, Ill.) or NOVABIOCHEM®. The aluminum chloridehexahydrate was purchased from SIGMA-ALDRICH® (Milwaukee, Wis.). Theremaining solvents and reagents were purchased from FISHER SCIENTIFIC®(Pittsburgh, Pa.) or SIGMA-ALDRICH® (Milwaukee, Wis.). ¹⁸F was suppliedby IBA MOLECULAR® (Somerset, N.J.)

¹⁸F-Labeling of IMP 272

The first peptide that was prepared and ¹⁸F-labeled was IMP 272:DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂ MH⁺ 1512

IMP 272 was synthesized as described (U.S. Pat. No. 7,534,431, theExamples section of which is incorporated herein by reference).

Acetate buffer solution—Acetic acid, 1.509 g was diluted in ˜160 mLwater and the pH was adjusted by the addition of 1 M NaOH then dilutedto 250 mL to make a 0.1 M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAcsolution at pH 4 to provide a 2 mM Al stock solution.

IMP 272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP 272was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

F-18 Labeling of IMP 272—A 3 μL aliquot of the aluminum stock solutionwas placed in a REACTI-VIAL™ and mixed with 50 μL ¹⁸F (as received) and3 μL of the IMP 272 solution. The solution was heated in a heating blockat 110° C. for 15 min and analyzed by reverse phase HPLC. HPLC analysis(not shown) showed 93% free ¹⁸F and 7% bound to the peptide. Anadditional 10 μL of the IMP 272 solution was added to the reaction andit was heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% ¹⁸F at the void volume and 92% of the activityattached to the peptide. The remainder of the peptide solution wasincubated at room temperature with 150 μL PBS for ˜1 hr and thenexamined by reverse phase HPLC. The HPLC (not shown) showed 58% ¹⁸Funbound and 42% still attached to the peptide. The data indicate that¹⁸F—Al-DTPA complex may be unstable when mixed with phosphate.

The labeled peptide was purified by applying the labeled peptidesolution onto a 1 cc (30 mg) WATERS® HLB column (Part #186001879) andwashing with 300 μL water to remove unbound F-18. The peptide was elutedby washing the column with 2×100 μL 1:1 EtOH/H₂O. The purified peptidewas incubated in water at 25° C. and analyzed by reverse phase HPLC (notshown). The HPLC analysis showed that the ¹⁸F-labeled IMP 272 was notstable in water. After 40 min incubation in water about 17% of the ¹⁸Fwas released from the peptide, while 83% was retained (not shown).

The peptide (16 μL 2 mM IMP 272, 48 μg) was labeled with ¹⁸F andanalyzed for antibody binding by size exclusion HPLC. The size exclusionHPLC showed that the peptide bound hMN-14×679 but did not bind to theirrelevant bispecific antibody hMN-14×734 (not shown).

IMP 272 ¹⁸F Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10⁻⁹ mol) was placed in apolypropylene cone vial and mixed with 75 μL ¹⁸F (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁻⁸ mol) IMP 272 solution in 0.1 M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown). These results demonstrate that the ¹⁸F metal labeling techniqueis not limited to an aluminum ligand, but can also utilize other metalsas well. With different metal ligands, different chelating moieties maybe utilized to optimize binding of an F-18-metal conjugate.

Production and Use of a Serum-Stable ¹⁸F-Labeled Peptide IMP 449

The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺ 1009 wasmade on Sieber Amide resin by adding the following amino acids to theresin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc wascleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make the desiredpeptide. The peptide was then cleaved from the resin and purified byHPLC to produce IMP 448, which was then coupled to ITC-benzyl NOTA. Thepeptide, IMP 448, 0.0757 g (7.5×10⁻⁵ mol) was mixed with 0.0509 g(9.09×10⁻⁵ mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassiumcarbonate anhydrous (0.2171 g) was then slowly added to the stirredpeptide/NOTA solution. The reaction solution was pH 10.6 after theaddition of all the carbonate. The reaction was allowed to stir at roomtemperature overnight. The reaction was carefully quenched with 1 M HClafter 14 hr and purified by HPLC to obtain 48 mg of IMP 449.

¹⁸F Labeling of IMP 449

The peptide IMP 449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2mM peptide solution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mMsolution of Al in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of¹⁸F. The solution was then mixed with 20 μL of the 2 mM IMP 449 solutionand heated at 105° C. for 15 min. Reverse Phase HPLC analysis showed 35%(t_(R)˜10 min) of the activity was attached to the peptide and 65% ofthe activity was eluted at the void volume of the column (3.1 min, notshown) indicating that the majority of activity was not associated withthe peptide. The crude labeled mixture (5 μL) was mixed with pooledhuman serum and incubated at 37° C. An aliquot was removed after 15 minand analyzed by HPLC. The HPLC showed 9.8% of the activity was stillattached to the peptide (down from 35%). Another aliquot was removedafter 1 hr and analyzed by HPLC. The HPLC showed 7.6% of the activitywas still attached to the peptide (down from 35%), which was essentiallythe same as the 15 min trace (data not shown).

High Dose ¹⁸F Labeling

Further studies with purified IMP 449 demonstrated that the ¹⁸F-labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. Additional studies wereperformed in which the IMP 449 was prepared in the presence of ascorbicacid as a stabilizing agent. In those studies (not shown), themetal-¹⁸F-peptide complex showed no detectable decomposition in serumafter 4 hr at 37° C. The mouse urine 30 min after injection of¹⁸F-labeled peptide was found to contain ¹⁸F bound to the peptide (notshown). These results demonstrate that the ¹⁸F-labeled peptidesdisclosed herein exhibit sufficient stability under approximated in vivoconditions to be used for ¹⁸F imaging studies.

For studies in the absence of ascorbic acid, ¹⁸F˜21 mCi in ˜400 μL ofwater was mixed with 9 μL of 2 mM AlCl₃ in 0.1 M pH 4 NaOAc. Thepeptide, IMP 449, 60 μL (0.01 M, 6×10⁻⁷ mol in 0.5 NaOH pH 4.13) wasadded and the solution was heated to 110° C. for 15 min. The crudelabeled peptide was then purified by placing the reaction solution inthe barrel of a 1 cc WATERS® HLB column and eluting with water to removeunbound ¹⁸F followed by 1:1 EtOH/H₂O to elute the ¹⁸F-labeled peptide.The crude reaction solution was pulled through the column into a wastevial and the column was washed with 3×1 mL fractions of water (18.97mCi). The HLB column was then placed on a new vial and eluted with 2×200μL 1:1 EtOH/H₂O to collect the labeled peptide (1.83 mCi). The columnretained 0.1 mCi of activity after all of the elutions were complete. Analiquot of the purified ¹⁸F-labeled peptide (20 μL) was mixed with 200μL of pooled human serum and heated at 37° C. Aliquots were analyzed byreverse phase HPLC. The results showed the relative stability of¹⁸F-labeled purified IMP 449 at 37° C. at time zero, one hour (91%labeled peptide), two hours (77% labeled peptide) and four hours (76%labeled peptide) of incubation in human serum (not shown). It was alsoobserved that ¹⁸F-labeled IMP 449 was stable in TFA solution, which isoccasionally used during reverse phase HPLC chromatography. Thereappears to be a general correlation between stability in TFA andstability in human serum observed for the exemplary ¹⁸F-labeledmolecules described herein. These results demonstrate that ¹⁸F-labeledpeptide, produced according to the methods disclosed herein, showssufficient stability in human serum to be successfully used for in vivolabeling and imaging studies, for example using PET scanning to detectlabeled cells or tissues. Finally, since IMP 449 peptide contains athiourea linkage, which is sensitive to radiolysis, several products areobserved by RP-HPLC. However, when ascorbic acid is added to thereaction mixture, the side products generated were markedly reduced.

Example 22 In Vivo Studies with Pretargeting TF10 DNL™ Construct and¹⁸F-Labeled Peptide

¹⁸F-labeled IMP 449 was prepared as follows. The ¹⁸F, 54.7 mCi in ˜0.5mL was mixed with 3 μL 2 mM Al in 0.1 M NaOAc pH 4 buffer. After 3 min10 μL of 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer was added and thereaction was heated in a 96° C. heating block for 15 min. The contentsof the reaction were removed with a syringe. The crude labeled peptidewas then purified by HPLC on a C₁₈ column. The flow rate was 3 mL/min.Buffer A was 0.1% TFA in water and Buffer B was 90% acetonitrile inwater with 0.1% TFA. The gradient went from 100% A to 75/25 A:B over 15min. There was about 1 min difference in retention time (t_(R)) betweenthe labeled peptide, which eluted first and the unlabeled peptide. TheHPLC eluent was collected in 0.5 min (mL) fractions. The labeled peptidehad a t_(R) between 6 to 9 min depending on the column used. The HPLCpurified peptide sample was further processed by diluting the fractionsof interest two fold in water and placing the solution in the barrel ofa 1 cc WATERS® HLB column. The cartridge was eluted with 3×1 mL water toremove acetonitrile and TFA followed by 400 μL 1:1 EtOH/H₂O to elute the¹⁸F-labeled peptide. The purified [Al¹⁸F] IMP 449 eluted as a singlepeak on an analytical HPLC C₁₈ column (not shown).

TACONIC® nude mice bearing the four slow-growing sc CaPan1 xenograftswere used for in vivo studies. Three of the mice were injected with TF10(162 μg) followed with [Al¹⁸F] IMP 449 18 h later. TF10 is a humanizedbispecific antibody of use for tumor imaging studies, with divalentbinding to the PAM-4 defined tumor antigen and monovalent binding to HSG(see, e.g., Gold et al., 2007, J. Clin. Oncol. 25(18S):4564). One mousewas injected with peptide alone. All of the mice were necropsied at 1 hpost peptide injection. Tissues were counted immediately. Comparison ofmean distributions showed substantially higher levels of ¹⁸F-labeledpeptide localized in the tumor than in any normal tissues in thepresence of tumor-targeting bispecific antibody (data not shown).

Tissue uptake was similar in animals given the [Al¹⁸F] IMP 449 alone orin a pretargeting setting (data not shown). Uptake in the humanpancreatic cancer xenograft, CaPan1, at 1 h was increased 5-fold in thepretargeted animals as compared to the peptide alone (4.6±0.9% ID/g vs.0.89% ID/g). Exceptional tumor/nontumor ratios were achieved at thistime (e.g., tumor/blood and liver ratios were 23.4±2.0 and 23.5±2.8,respectively).

The results demonstrate that ¹⁸F labeled peptide used in conjunctionwith a PAM4 containing antibody construct, such the TF10 DNL™ construct,provide suitable targeting of the ¹⁸F label to perform in vivo imaging,such as PET imaging analysis.

Example 23 Further Imaging Studies with TF10

Summary

Preclinical and clinical studies have demonstrated the application ofradiolabeled mAb-PAM4 for nuclear imaging and radioimmunotherapy ofpancreatic carcinoma. We have examined herein the ability of the TF10construct to pretarget a radiolabeled peptide for improved imaging andtherapy. Biodistribution studies and nuclear imaging of the radiolabeledTF10 and/or TF10-pretargeted hapten-peptide (IMP-288) were conducted innude mice bearing CaPan1 human pancreatic cancer xenografts. ¹²⁵I-TF10cleared rapidly from the blood, with levels decreasing to <1% injecteddose per gram (ID/g) by 16 hours. Tumor uptake was 3.47±0.66% ID/g atthis time point with no accumulation in any normal tissue. To show theutility of the pretargeting approach, ¹¹¹In-IMP-288 was administered 16hours after TF10. At 3 hours postadministration of radiolabeled peptide,imaging showed intense uptake within the tumors and no evidence ofaccretion in any normal tissue. No targeting was observed in animalsgiven only the ¹¹¹In-peptide. Tumor uptake of the TF10-pretargeted¹¹¹In-IMP-288 was 24.3±1.7% ID/g, whereas for ¹¹¹In-IMP-288 alone it wasonly 0.12±0.002% ID/g at 16 hours. Tumor/blood ratios were significantlygreater for the pretargeting group (˜1,000:1 at 3 hours) compared with¹¹¹In-PAM4-IgG (˜5:1 at 24 hours; P<0.0003). Radiation dose estimatessuggested that TF10/⁹⁰Y-peptide pretargeting would provide a greaterantitumor effect than ⁹⁰Y-PAM4-IgG. Thus, the results support that TF10pretargeting may provide improved imaging for early detection,diagnosis, and treatment of pancreatic cancer as compared with directlyradiolabeled PAM4-IgG. (Gold et al., Cancer Res 2008, 68(12):4819-26)

We have identified a unique biomarker present on mucin expressed by >85%of invasive pancreatic adenocarcinomas, including early stage I diseaseand the precursor lesions, pancreatic intraepithelial neoplasia andintraductal papillary mucinous neoplasia (Gold et al., Clin Cancer Res2007, 13:7380-87). The specific epitope, as detected by mAb-PAM4 (Goldet al., Int J Cancer 1994, 57:204-10), is absent from normal andinflamed pancreatic tissues, as well as most other malignant tissues.Thus, detection of the epitope provides a high diagnostic likelihood forthe presence of pancreatic neoplasia. Early clinical studies using ¹³¹I-and ^(99m)Tc-labeled, murine PAM4 IgG or Fab′, respectively, showedspecific targeting in 8 of 10 patients with invasive pancreaticadenocarcinoma (Mariani et al., Cancer Res 1995, 55:5911s-15s; Gold etal., Crit Rev Oncol Hematol 2001, 39:147-54). Of the two negativepatients, one had a poorly differentiated pancreatic carcinoma that didnot express the PAM4-epitope, whereas the other patient was later foundto have pancreatitis rather than a malignant lesion.

Accordingly, the high specificity of PAM4 for pancreatic cancer is ofuse for the detection and diagnosis of early disease. In addition toimproved detection, ⁹⁰Y-PAM4 IgG was found to be effective in treatinglarge human pancreatic cancer xenografts in nude mice (Cardillo et al.,Clin Cancer Res 2001, 7:3186-92), and when combined with gemcitabine,further improvements in therapeutic response were observed (Gold et al.,Clin Cancer Res 2004, 10:3552-61; Gold et al., Int J Cancer 2004,109:618-26). A Phase I therapy trial in patients who failed gemcitabinetreatment was recently completed, finding the maximum tolerated dose of⁹⁰Y-humanized PAM4 IgG to be 20 mCi/m² (Gulec et al., Proc Amer Soc ClinOnc, 43rd Annual Meeting, J Clin Oncol 2007, 25(18S):636s). Although allpatients showed disease progression at or after week 8, initialshrinkage of tumor was observed in several cases. Clinical studies arenow underway to evaluate a fractionated dosing regimen of ⁹⁰Y-hPAM4 IgGin combination with a radiosensitizing dose of gemcitabine.

We report herein the development of a novel recombinant, humanizedbispecific monoclonal antibody (mAb), TF10, based on the targetingspecificity of PAM4 to pancreatic cancer. This construct also binds tothe unique synthetic hapten, histamine-succinyl-glycine (HSG), which hasbeen incorporated in a number of small peptides that can be radiolabeledwith a wide range of radionuclides suitable for single-photon emissioncomputed tomography (SPECT) and positron emission tomography (PET)imaging, as well as for therapeutic purposes (Karacay et al., ClinCancer Res 2005, 11:7879-85; Sharkey et al., Leukemia 2005, 19:1064-9;Rossi et al., Proc Natl Acad Sci USA 2006, 103:6841-6; McBride et al., JNucl Med 2006, 47:1678-88). These studies illustrate the potential ofthis new construct to target pancreatic adenocarcinoma for imaging ortherapeutic applications.

Methods and Materials

The TF2 and TF10 bispecific DNL™ constructs and the IMP 288 targetingpeptide were prepared as described above. Sodium iodide (¹²⁵I) andindium chloride (¹¹¹In) were obtained from PERKIN-ELMER®. TF10 wasroutinely labeled with ¹²⁵I by the iodogen method, with purification byuse of size-exclusion spin columns. Radiolabeling of DOTA-peptide andDOTA-PAM4-IgG with ¹¹¹InCl was done as previously described (Rossi etal., Proc Natl Acad Sci USA 2006, 103:6841-6; McBride et al., J Nucl Med2006, 47:1678-88). Purity of the radiolabeled products was examined bysize-exclusion high-performance liquid chromatography with the amount offree, unbound isotope determined by instant TLC.

For TF10 distribution studies, female athymic nude mice ˜20 g (TACONIC®Farms), bearing s.c. CaPan1 human pancreatic cancer xenografts, wereinjected with ¹²⁵I-TF10 (10 μCi; 40 μg, 2.50×10⁻¹⁰ mol). At various timepoints, groups of mice (n=5) were necropsied, with tumor and nontumortissues removed and counted in a gamma counter to determine thepercentage of injected dose per gram of tissue (% ID/g), with thesevalues used to calculate blood clearance rates and tumor/nontumorratios.

For pretargeting biodistribution studies, a bispecific mAb/radiolabeledpeptide molar ratio of 10:1 was used. For example, a group of athymicnude mice bearing s.c. CaPan1 human pancreatic cancer xenografts wasadministered TF10 (80 μg, 5.07×10⁻¹⁰ mol), whereas a second group wasleft untreated. At 16 h postinjection of TF10, ¹¹¹In-IMP-288hapten-peptide (30 μCi, 5.07×10⁻¹¹ mol) was administered. Mice werenecropsied at several time points, with tumor and nontumor tissuesremoved and counted in a gamma counter to determine the % ID/g.Tumor/nontumor ratios were calculated from these data. In a separatestudy, groups of mice were given ¹¹¹In-DOTA-PAM4-IgG (20 μC, 50 μg,3.13×10⁻¹⁰ mol) for the purpose of comparing biodistribution, nuclearimaging, and potential therapeutic activity. Radiation dose estimateswere calculated from the time-activity curves with the assumption of noactivity at zero time. Student's t test was used to assess significantdifferences.

To perform nuclear immunoscintigraphy, at 3 h postinjection ofradiolabeled peptide or 24 h postinjection of radiolabeled hPAM4-IgG,tumor-bearing mice were imaged with a dual-head Solus gamma camerafitted with medium energy collimator for ¹¹¹In (ADAC Laboratories). Micewere imaged for a total of 100,000 cpm or 10 min, whichever came first.

Results

In Vitro Characterization of the Bispecific mAb TF10.

The binding of TF10 to the target mucin antigen was analyzed by ELISA(FIG. 16). The results showed nearly identical binding curves for thedivalent TF10, PAM4-IgG, and PAM4-F(ab′)₂ (half-maximal binding wascalculated as 1.42±0.10, 1.31±0.12, and 1.83±0.16 nmol/L, respectively;P>0.05 for all), whereas the monovalent bsPAM4 chemical conjugate(PAM4-Fab′×anti-DTPA-Fab′) had a significantly lower avidity(half-maximal binding, 30.61±2.05 nmol/L; P=0.0379, compared with TF10),suggesting that TF10 binds in a divalent manner. The immunoreactivefraction of ¹²⁵I-TF10 bound to MUC5AC was 87%, with 9% found as unboundTF10 and 3% as free iodide (not shown). Ninety percent of the¹¹¹In-IMP-288 bound to TF10 (not shown). Of the total ¹¹¹In-IMP-288bound to TF10, 92% eluted at higher molecular weight when excess mucin(200 μg) was added, with only 3% eluting with the non-mucin-reactiveTF10 fraction. An additional 5% of the radiolabeled peptide eluted inthe free peptide volume. None of the radiolabeled peptide bound to themucin antigen in the absence of TF10 (not shown).

Biodistribution of ¹²⁵I-TF10 in CaPan1 Tumor-Bearing Nude Mice.

TF10 showed a rapid clearance from the blood, starting with 21.03±1.93%ID/g at 1 hour and decreasing to just 0.13±0.02% ID/g at 16 hours. Thebiological half-life was calculated to be 2.19 hours [95% confidenceinterval (95% CI), 2.11-2.27 hours]. Tissue uptake revealed enhancedactivity in the liver, spleen, and kidneys at 1 hour, which cleared justas quickly by 16 hours [T_(1/2)=2.09 hours (95% CI, 2.08-2.10), 2.84hours (95% CI, 2.49-3.29), and 2.44 hours (95% CI, 2.28-2.63) for liver,spleen, and kidney, respectively]. Activity in the stomach most likelyreflects the accretion and excretion of radioiodine, suggesting that theradioiodinated TF10 was actively catabolized, presumably in the liverand spleen, thereby explaining its rapid clearance from the blood.Nevertheless, by 16 hours, the concentration of radioiodine within thestomach was below 1% ID/g. A group of five non-tumor-bearing nude micegiven ¹²⁵I-TF10 and necropsied at 16 hours showed similar tissuedistribution, suggesting that the tumor had not affected the bispecificmAb distribution and clearance from normal tissues (data not shown). Ofcourse, it is possible that differences occurred before the initial timepoint examined. Tumor uptake of TF10 peaked at 6 hours postinjection(7.16±1.10% ID/g) and had decreased to half maximum binding (3.47±0.66%ID/g) at 16 hours. Tumor uptake again decreased nearly 2-fold over thenext 32 hours, but then was stable over the following 24 hours.

Biodistribution of TF10-Pretargeted, ¹¹¹In-Labeled Peptide.

Although maximum tumor uptake of TF10 occurred at 6 hours, previousexperience indicated that the radiolabeled peptide would need to begiven at a time point when blood levels of TF10 had cleared to <1% ID/g(i.e., 16 hours). Higher levels of TF10 in the blood would lead tounacceptably high binding of the radiolabeled peptide within the blood(i.e., low tumor/blood ratios), whereas administering the peptide at alater time would mean the concentration of TF10 in the tumor would bedecreased with consequently reduced concentration of radiolabeledpeptide within the tumor. Thus, an initial pretargeting study was doneusing a 16-hour interval. With the amount of the ¹¹¹In-IMP-288 heldconstant (30 μCi, 5.07×10⁻¹¹ mol), increasing amounts of TF10 were givenso that the administered dose of TF10 and IMP-288 expressed as moleratio varied from 5:1 to 20:1 (Table 16).

TABLE 16 Biodistribution of ¹¹¹In-IMP-288 alone (no TF10) or pretargetedwith varying amounts of TF10 % ID/g at 3 h (mean ± SD) 5:1 10:1 20:1 NoTF10 Tumor 19.0 ± 3.49 24.3 ± 1.71 28.6 ± 0.73 0.12 ± 0.00 Liver 0.09 ±0.01 0.21 ± 0.12 0.17 ± 0.01 0.07 ± 0.00 Spleen 0.12 ± 0.04 0.16 ± 0.070.26 ± 0.10 0.04 ± 0.01 Kidneys 1.59 ± 0.11 1.72 ± 0.24 1.53 ± 0.14 1.71± 0.22 Lungs 0.19 ± 0.06 0.26 ± 0.00 0.29 ± 0.04 0.03 ± 0.00 Blood 0.01± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 Stomach 0.03 ± 0.02 0.02 ±0.02 0.01 ± 0.00 0.02 ± 0.01 Small intestine 0.12 ± 0.08 0.08 ± 0.030.04 ± 0.01 0.06 ± 0.02 Large intestine 0.23 ± 0.10 0.39 ± 0.08 0.25 ±0.08 0.33 ± 0.02 Pancreas 0.02 ± 0.00 0.02 ± 0.01 0.02 ± 0.00 0.02 ±0.00 Tumor 0.12 ± 0.03 0.32 ± 0.09 0.27 ± 0.01 0.35 ± 0.03 weight (g)

At 3 hours the amount of ¹¹¹In-IMP-288 in the blood was barelydetectable (0.01%). Tumor uptake increased from 19.0±3.49% ID/g to28.55±0.73% ID/g as the amount of bispecific mAb administered wasincreased 4-fold (statistically significant differences were observedfor comparison of each TF10/peptide ratio, one group to another; P<0.03or better), but without any appreciable increase in normal tissueuptake. Tumor uptake in the animals given TF10 was >100-fold higher thanwhen ¹¹¹In-IMP-288 was given alone. Comparison of ¹¹¹In activity in thenormal tissues of the animals that either received or did not receiveprior administration of TF10 indicated similar absolute values, which inmost instances were not significantly different. This suggests that thebispecific mAb had cleared sufficiently from all normal tissues by 16hours to avoid appreciable peptide uptake in these tissues. Tumor/bloodratios were >2,000:1, with other tissue ratios exceeding 100:1. Eventumor/kidney ratios exceeded 10:1. The highest tumor uptake ofradioisotope with minimal targeting to nontumor tissues resulted fromthe 20:1 ratio; however, either of the TF10/peptide ratios could be usedto achieve exceptional targeting to tumor, both in terms of signalintensity and contrast ratios. The 10:1 ratio was chosen for furtherstudy because the absolute difference in tumor uptake of radiolabeledpeptide was not substantially different between the 10:1 (24.3±1.71%ID/g) and 20:1 (28.6±0.73% ID/g) ratios.

Images of the animals given TF10-pretargeted ¹¹¹In-IMP-288 at abispecific mAb/peptide ratio of 10:1, or the ¹¹¹In-IMP-288 peptidealone, are shown in FIG. 17A, FIG. 17B and FIG. 17C. The majority ofthese tumors were <0.5 cm in diameter, weighing ˜0.25 g. The images showhighly intense uptake in the tumor of the TF10-pretargeted animals (FIG.17A). The intensity of the image background for the TF10-pretargetedanimals was increased to match the intensity of the image taken of theanimals given the ¹¹¹In-IMP-288 alone (FIG. 17B). However, when theimages were optimized for the TF10-pretargeted mice, the signalintensity and contrast were so high that no additional activity wasobserved in the body. No tumor localization was seen in the animalsgiven the ¹¹¹In-IMP-288 alone, even when image intensity was enhanced(FIG. 17C).

An additional experiment was done to assess the kinetics of targeting¹¹¹In-hPAM4 whole-IgG compared with that of the TF10-pretargeted¹¹¹In-IMP-288 peptide. Tumor uptake of the ¹¹¹In-peptide was highest atthe initial time point examined, 3 hours (15.99±4.11% ID/g), whereas theblood concentration of radiolabeled peptide was only 0.02±0.01% ID/g,providing a mean tumor/blood ratio of 946.3±383.0. Over time,radiolabeled peptide cleared from the tumor with a biological half-lifeof 76.04 hours. Among nontumor tissues, uptake was highest in thekidneys, averaging 1.89±0.42% ID/g at 3 hours with a steady decreaseover time (biological half-life, 33.6 hours). Liver uptake started at0.15±0.06% ID/g and remained essentially unchanged over time. Incontrast to the TF10-pretargeted ¹¹¹In-IMP-288, the ¹¹¹In-hPAM4-IgG hada slower clearance from the blood, albeit there was a substantialclearance within the first 24 hours, decreasing from 30.1% ID/g at 3hours to just 11.5±1.7% ID/g at 24 hours. Variable elevated uptake inthe spleen suggested that the antibody was likely being removed from theblood by targeting of secreted mucin that had become entrapped withinthe spleen. Tumor uptake peaked at 48 hours with 80.4±6.1% ID/g, andremained at an elevated level over the duration of the monitoringperiod. The high tumor uptake, paired with a more rapid than expectedblood clearance for an IgG, produced tumor/blood ratios of 5.2±1.0within 24 hours. FIG. 17C shows the images of the animals at 24 hourspostadministration of ¹¹¹In-PAM4-IgG, illustrating that tumors could bevisualized at this early time, but there was still considerable activitywithin the abdomen. Tumor/nontumor ratios were mostly higher forTF10-pretargeted ¹¹¹In-labeled hapten-peptide as compared with¹¹¹In-hPAM4-IgG, except for the kidneys, where tumor/kidney ratios withthe ¹¹¹In-IMP-288 and ¹¹¹In-hPAM4-IgG were similar at later times.However, tumor/kidney ratios for the TF10-pretargeted ¹¹¹In-IMP-288 werehigh enough (e.g., ˜7:1) at 3 hours to easily discern tumor from normaltissue.

FIG. 18A to FIG. 18D illustrates the potential therapeutic capability ofthe direct and pretargeted methods to deliver radionuclide (⁹⁰Y).Although the concentration (% ID/g) of radioisotope within the tumorseems to be much greater when delivered by PAM4-IgG than by pretargetedTF10 at their respective maximum tolerated dose (0.15 mCi for ⁹⁰Y-hPAM4and 0.9 mCi for TF10-pretargeted ⁹⁰Y-IMP-288) (FIG. 18A), the radiationdose to tumor would be similar (10,080 and 9,229 cGy for ⁹⁰Y-PAM4-IgGand TF10-pretargeted ⁹⁰Y-IMP-288, respectively) (FIG. 18C). Theadvantage for the pretargeting method would be the exceptionally lowactivity in blood (9 cGy), almost 200-fold less than with the ⁹⁰Y-hPAM4IgG (1,623 cGy) (FIG. 18C). It is also important to note that theradiation dose to liver, as well as other nontumor organs, would be muchlower with the TF10-pretargeted ⁹⁰Y-IMP-288 (FIG. 18B, FIG. 18D).

The exception would be the kidneys, where the radiation dose would besimilar for both protocols at their respective maximum dose (612 and 784cGy for ⁹⁰Y-PAM4-IgG and TF10-⁹⁰Y-IMP-288, respectively) (FIG. 18B, FIG.18D). The data suggest that for ⁹⁰Y-PAM4-IgG, as with most otherradiolabeled whole-IgG mAbs, the dose-limiting toxicity would behematologic; however, for the TF10 pretargeting protocol, thedose-limiting toxicity would be the kidneys.

Discussion

Current diagnostic modalities such as ultrasound, computerizedtomography (CT), and magnetic resonance imaging (MRI) technologies,which provide anatomic images, along with PET imaging of the metabolicenvironment, have routinely been found to provide high sensitivity inthe detection of pancreatic masses. However, these data are, for themost part, based on detection of lesions >2 cm in a population that isalready presenting clinical symptoms. At this time in the progression ofthe pancreatic carcinoma, the prognosis is rather dismal. To improvepatient outcomes, detection of small, early pancreatic neoplasms in theasymptomatic patient is necessary.

Imaging with a mAb-targeted approach, such as is described herein withmAb-PAM4, may provide for the diagnosis of these small, early cancers.Of prime importance is the specificity of the mAb. We have presentedconsiderable data, including immunohistochemical studies of tissuespecimens (Gold et al., Clin Cancer Res 2007; 13:7380-7; Gold et al.,Int J Cancer 1994; 57:204-10) and immunoassay of patient sera (Gold etal., J Clin Oncol 2006; 24:252-8), to show that mAb-PAM4 is highlyreactive with a biomarker, the presence of which provides highdiagnostic likelihood of pancreatic neoplasia. Furthermore, wedetermined that PAM4, although not reactive with normal adult pancreatictissues nor active pancreatitis, is reactive with the earliest stages ofneoplastic progression within the pancreas (pancreatic intraepithelialneoplasia 1 and intraductal papillary mucinous neoplasia) and that thebiomarker remains at high levels of expression throughout theprogression to invasive pancreatic adenocarcinoma (Gold et al., ClinCancer Res 2007; 13:7380-7). Preclinical studies with athymic nude micebearing human pancreatic tumor xenografts have shown specific targetingof radiolabeled murine, chimeric, and humanized versions of PAM4.

In the current studies, we have examined a next-generation, recombinant,bispecific PAM4-based construct, TF10, which is divalent for the PAM4arm and monovalent for the anti-HSG hapten arm. There are severalimportant characteristics of this pretargeting system's constructs,named DOCK-AND-LOCK™, including its general applicability and ease ofsynthesis. However, for the present consideration, the major differencesfrom the previously reported chemical construct are the valency, whichprovides improved binding to tumor antigen, and, importantly, itspharmacokinetics. TF10 clearance from nontumor tissues is much morerapid than was observed for the chemical conjugate. Time for bloodlevels of the bispecific constructs to reach less than 1% ID/g was 40hours postinjection for the chemical construct versus 16 hours for TF10.A more rapid clearance of the pretargeting agent has provided a vastimprovement of the tumor/blood ratio, while maintaining high signalstrength at the tumor site (% ID/g).

In addition to providing a means for early detection and diagnosis, theresults support the use of the TF10 pretargeting system for cancertherapy. Consideration of the effective radiation dose to tumor andnontumor tissues favors the pretargeting method over directlyradiolabeled PAM4-IgG. The dose estimates suggest that the two deliverysystems have different dose-limiting toxicities: myelotoxicity for thedirectly radiolabeled PAM4 versus the kidney for the TF10 pretargetingsystem. This is of significance for the future clinical development ofradiolabeled PAM4 as a therapeutic agent.

Gemcitabine, the frontline drug of choice for pancreatic cancer, canprovide significant radiosensitization of tumor cells. In previousstudies, we showed that combinations of gemcitabine and directlyradiolabeled PAM4-IgG provided synergistic antitumor effects comparedwith either arm alone (Gold et al., Clin Cancer Res 2004, 10:3552-61;Gold et al., Int J Cancer 2004, 109:618-26). The dose-limiting factorwith this combination was overlapping hematologic toxicity. However,because the dose-limiting organ for TF10 pretargeting seems to be thekidney rather than hematologic tissues, combinations with gemcitabineshould be less toxic, thus allowing increased administration ofradioisotope with consequently greater antitumor efficacy.

The superior imaging achieved with TF10 pretargeting in preclinicalmodels, as compared with directly radiolabeled DOTA-PAM4-IgG, provides acompelling rationale to proceed to clinical trials with this imagingsystem. The specificity of the tumor-targeting mAb for pancreaticneoplasms, coupled with the bispecific antibody platform technologyproviding the ability to conjugate various imaging compounds to theHSG-hapten-peptide for SPECT (¹¹¹In), PET (⁶⁸Ga), ultrasound (Au), orother contrast agents, or for that matter ⁹⁰Y or other radionuclides fortherapy, provides high potential to improve overall patient outcomes(Goldenberg et al., J Nucl Med 2008, 49:158-63). In particular, webelieve that a TF10-based ImmunoPET procedure will have major clinicalvalue to screen individuals at high-risk for development of pancreaticcancer (e.g., genetic predisposition, chronic pancreatitis, smokers,etc.), as well as a means for follow-up of patients with suspiciousabdominal images from conventional technologies and/or with indicationsdue to the presence of specific biomarker(s) or abnormal biochemicalfindings. When used as part of an ongoing medical plan for followingthese patients, early detection of pancreatic cancer may be achieved.Finally, in combination with gemcitabine, TF10 pretargeting may providea better opportunity for control of tumor growth than directlyradiolabeled PAM4-IgG.

Example 24 Therapy of Pancreatic Cancer Xenografts with Gemcitabine and⁹⁰Y-Labeled Peptide Pretargeted Using TF10 Summary

⁹⁰Y-hPAM4 IgG is currently being examined in Phase I/II trials incombination with gemcitabine in patients with Stage III/IV pancreaticcancer. We disclose a new approach for pretargeting radionuclides thatis able to deliver a similar amount of radioactivity to pancreaticcancer xenografts, but with less hematological toxicity, which would bemore amenable for combination with gemcitabine. Nude mice bearing ˜0.4cm³ sc CaPan1 human pancreatic cancer were administered a recombinantbsMAb, TF10, followed 1 day later with a ⁹⁰Y-labeled hapten-peptide(IMP-288). Various doses and schedules of gemcitabine were added to thistreatment, and tumor progression monitored up to 28 weeks. 0.7 mCiPT-RAIT alone produce only a transient 60% loss in blood counts, andanimals given 0.9 mCi of PT-RAIT alone and 0.7 mCi PT-RAIT+6 mggemcitabine (human equivalent ˜1000 mg/m²) had no histological evidenceof renal toxicity after 9 months. A single dose of 0.25 or 0.5 mCiPT-RAIT alone can completely ablate 20% and 80% of the tumors,respectively. Monthly fractionated PT-RAIT (0.25 mCi/dose given at thestart of each gemcitabine cycle) added to a standard gemcitabine regimen(6 mg wkly×3; 1 wk off; repeat 3 times) significantly increased themedian time for tumors to reach 3.0 cm³ over PT-RAIT alone. Othertreatment plans examining non-cytotoxic radiosensitizing dose regimensof gemcitabine added to PT-RAIT also showed significant improvements intreatment response over PT-RAIT alone. The results show that PT-RAIT isa promising new approach for treating pancreatic cancer. Current dataindicate combining PT-RAIT with gemcitabine will enhance therapeuticresponses.

Methods

TF10 bispecific antibody was prepared as described above. Forpretargeting, TF10 was given to nude mice bearing the human pancreaticadenocarcinoma cell line, CaPan1. After allowing sufficient time forTF10 to clear from the blood (16 h), the radiolabeled divalentHSG-peptide was administered. The small molecular weight HSG-peptide(˜1.4 kD) clears within minutes from the blood, entering theextravascular space where it can bind to anti-HSG arm of the pretargetedTF10 bsMAb. Within a few hours, >80% of the radiolabeled HSG-peptide isexcreted in the urine, leaving the tumor localized peptide and a traceamount in the normal tissues.

Results

FIG. 19 illustrates the therapeutic activity derived from a singletreatment of established (˜0.4 cm³) CaPan1 tumors with 0.15 mCi of⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCi of TF10-pretargeted ⁹⁰Y-IMP-288.Similar anti-tumor activity was observed for the 0.5-mCi pretargeteddose vs. 0.15-mCi dose of the directly radiolabeled IgG, buthematological toxicity was severe at this level of the direct conjugate(not shown), while the pretargeted dose was only moderately toxic (notshown). Indeed, the MTD for pretargeting using 90Y-IMP-288 is at least0.9 mCi in nude mice.

FIG. 20 shows that the combination of gemcitabine and PT-RAIT has asynergistic effect on anti-tumor therapy. Human equivalent doses of 1000mg/m² (6 mg) of gemcitabine (GEM) were given intraperitoneally to miceweekly for 3 weeks, then after resting for 1 week, this regimen wasrepeated 2 twice. PT-RAIT (0.25 mCi of TF10-pretargeted ⁹⁰Y-IMP-288) wasgiven 1 day after the first GEM dose in each of the 3 cycles oftreatment. Gem alone had no significant impact on tumor progression(survival based on time to progress to 3.0 cm³). PT-RAIT alone improvedsurvival compared to untreated animals, but the combined GEM withPT-RAIT regimen increased the median survival by nearly 10 weeks.Because hematological toxicity is NOT dose-limiting for PT-RAIT, but itis one of the limitations for gemcitabine therapy, these studies suggestthat PT-RAIT could be added to a standard GEM therapy with the potentialfor enhanced responses. The significant synergistic effect ofgemcitabine plus PT-RAIT was surprising and unexpected.

A further study examined the effect of the timing of administration onthe potentiation of anti-tumor effect of gemcitabine plus PT-RAIT. Asingle 6-mg dose of GEM was given one day before or 1 day after 0.25 mCiof TF10-pretargeted ⁹⁰Y-IMP-288 (not shown). This study confirmed whatis already well known with GEM, i.e., radiosensitization is best givenin advance of the radiation. Percent survival of treated mice showedlittle difference in survival time between PT-RAIT alone and PT-RAITwith gemcitabine given 22 hours after the radiolabeled peptide. However,administration of gemcitabine 19 hours prior to PT-RAIT resulted in asubstantial increase in survival (not shown).

Single PT-RAIT (0.25 mCi) combined with cetuximab (1 mg weekly ip; 7weeks) or with cetuximab+GEM (6 mg weekly×3) in animals bearing CaPan1showed the GEM+cetuximab combination with PT-RAIT providing a betterinitial response (FIG. 21), but the response associated with justcetuximab alone added to PT-RAIT was encouraging (FIG. 21), since it wasas good or better than PT-RAIT+GEM. Because the overall survival in thisstudy was excellent, with only 2 tumors in each group progressingto >2.0 cm3 after 24 weeks when the study was terminated, these resultsindicate a potential role for cetuximab when added to PT-RAIT.

Example 25 Effect of Fractionated Pretargeted Radioimmunotherapy(PT-RAIT) for Pancreatic Cancer Therapy

We evaluated fractionated therapy with ⁹⁰Y-DOTA-di-HSG peptide (IMP-288)and TF10. Studies using TF10 and radiolabeled IMP-288 were performed innude mice bearing s.c. CaPan1 human pancreatic cancer xenografts,0.32-0.54 cm³. For therapy, TF10-pretargeted ⁹⁰Y-IMP-288 was given [A]once (0.6 mCi on wk 0) or [B] fractionated (0.3 mCi on wks 0 and 1),[C](0.2 mCi on wks 0, 1 and 2) or [D] (0.2 mCi on wks 0, 1 and 4).

Tumor regression (>90%) was observed in the majority of mice, 9/10,10/10, 9/10 and 8/10 in groups [A], [B], [C] and [D], respectively. Ingroup [A], maximum tumor regression in 50% of the mice was reached at3.7 wks, compared to 6.1, 8.1 and 7.1 wks in [B], [C] and [D],respectively. Some tumors showed regrowth. At week 14, the besttherapeutic response was observed in the fractionated group (2×0.3 mCi),with 6/10 mice having no tumors (NT) compared to 3/10 in the 3×0.2 mCigroups and 1/10 in the 1×0.6 mCi group. No major body weight loss wasobserved. Fractionated PT-RAIT provides another alternative for treatingpancreatic cancer with minimum toxicity.

Example 26 ⁹⁰Y-hPAM4 Radioimmunotherapy (RAIT) Plus RadiosensitizingGemcitabine (GEM) Treatment in Advanced Pancreatic Cancer (PC)

⁹⁰Y-hPAM4, a humanized antibody highly specific for PC, showed transientactivity in patients with advanced disease, and GEM enhanced RAIT inpreclinical studies. This study evaluated repeated treatment cycles of⁹⁰Y-hPAM4 plus GEM in patients with untreated, unresectable PC. The⁹⁰Y-dose was escalated by cohort, with patients repeating 4-wk cycles(once weekly 200 mg/m² GEM, ⁹⁰Y-hPAM4 once-weekly wks 2-4) untilprogression or unacceptable toxicity. Response assessments used CT,FDG-PET, and CA19.9 serum levels.

Of 8 patients (3F/5M, 56-72 y.o.) at the 1^(st) 2 dose levels (6.5 and9.0 mCi/m² ⁹⁰Y-hPAM4×3), hematologic toxicity has been transient Grade1-2. Two patients responded to initial treatment with FDG SUV and CA19.9decreases, and lesion regression by CT. Both patients continue in goodperformance status now after 9 and 11 mo. and after a total of 3 and 4cycles, respectively, without additional toxicity. A 3^(rd) patient witha stable response by PET and CT and decreases in CA19.9 levels afterinitial treatment is now undergoing a 2nd cycle. Four other patients hadearly disease progression and the remaining patient is still beingevaluated. Dose escalation is continuing after fractionated RAIT with⁹⁰Y-hPAM4 plus low-dose gemcitabine demonstrated therapeutic activity atthe initial ⁹⁰Y-dose levels, with minimal hematologic toxicity, evenafter 4 therapy cycles.

Example 27 Early Detection of Pancreatic Carcinoma Using Mab-PAM4 and inVitro Immunoassay

Immunohistochemistry studies were performed with PAM4 antibody. Resultsobtained with stained tissue sections showed no reaction of PAM4 withnormal pancreatic ducts, ductules and acinar tissues (not shown). Incontrast, use of the MA5 antibody applied to the same tissue samplesshowed diffuse positive staining of normal pancreatic ducts and acinartissue (not shown). In tissue sections with well differentiated ormoderately differentiated pancreatic adenocarcinoma, PAM4 staining waspositive, with mostly cytoplasmic staining but intensification of at thecell surface. Normal pancreatic tissue in the same tissue sections wasunstained.

Table 17 shows the results of immunohistochemical analysis with PAM4 MAbin pancreatic adenocarcinoma samples of various stages ofdifferentiation. Overall, there was an 87% detection rate for allpancreatic cancer samples, with 100% detection of well differentiatedand almost 90% detection of moderately differentiated pancreaticcancers.

TABLE 17 PAM4 Labeling Pattern Cancer n Focal Diffuse Total Well Diff.13 2 11  13 (100%) Moderately Diff. 24 6 15 21 (88%) Poorly Diff. 18 5 914 (78%) Total 55 13 35 48 (87%)

Table 18 shows that PAM4 immunohistochemical staining also detected avery high percentage of precursor lesions of pancreatic cancer,including PanIn-1A to PanIN-3, IPMN (intraductal papillary mucinousneoplasms) and MCN (mucinous cystic neoplasms). Overall, PAM4 stainingdetected 89% of all pancreatic precursor lesions. These resultsdemonstrate that PAM4 antibody-based immunodetection is capable ofdetecting almost 90% of pancreatic cancers and precursor lesions by invitro analysis. PAM4 expression was observed in the earliest phases ofPanIN development. Intense staining was observed in IPMN and MCN samples(not shown). The PAM4 epitope was present at high concentrations(intense diffuse stain) in the great majority of pancreaticadenocarcinomas. PAM4 showed diffuse, intense reactivity with theearliest stages of pancreatic carcinoma precursor lesions, includingPanIN-1, IPMN and MCN, yet was non-reactive with normal pancreatictissue. Taken together, these results show that diagnosis and/ordetection with the PAM4 antibody is capable of detecting, with very highspecificity, the earliest stages of pancreatic cancer development.

TABLE 18 PAM4 Labeling Pattern n Focal Diffuse Total PanIn-1A 27 9 15 24(89%) PanIn-1B 20 4 16  20 (100%) PanIn-2 11 6 4 10 (91%) PanIn-3 5 2 0 2 (40%) Total PanIn 63 21 35 56 (89%) IPMN 36 6 25 31 (86%) MCN 27 3 2225 (92%)

An enzyme based immunoassay for PAM4 antigen in serum samples wasdeveloped. FIG. 22 shows the results of differential diagnosis usingPAM4 immunoassay for pancreatic cancer versus normal tissues and othertypes of cancer. The results showed a sensitivity of detection ofpancreatic cancer of 77.4%, with a specificity of detection of 94.3%,comparing pancreatic carcinoma (n=53) with all other specimens (n=233),including pancreatitis and breast, ovarian and colorectal cancer andlymphoma. The data of FIG. 22 are presented in tabular form in Table 19.

TABLE 19 PAM4-Reactive MUC5AC in Patient Sera # Positive n Mean SDMedian Range (%) Normal 43 0.1 0.3 0.0 0-2.0 0 (0) Pancreatitis 87 3.011.5 0.0 0-63.6 4 (5) Pancreatic CA 53 171 317 31.7 0-1000 41 (77)Colorectal CA 36 3.3 7.7 0.0 0-37.8  5 (14) Breast CA 30 3.7 10.1 0.00-53.5 2 (7) Ovarian CA 15 1.8 4.3 0.0 0-16.5 1 (7) Lymphoma 19 12.344.2 0.0 0-194 1 (5)

An ROC curve (not shown) was constructed with the data from Table 19.Examining a total of 283 patients, including 53 with pancreaticcarcinoma, and comparing the presence of circulating MUC5AC in patientswith pancreatic cancer to all other samples, the ROC curve provided anAUC of 0.88±0.03 (95% ci, 0.84-0.92) with a P value <0.0001, a highlysignificant difference for discrimination of pancreatic carcinoma fromnon-pancreatic carcinoma samples. Comparing pancreatic CA with othertumors and normal tissue, the PAM4 based serum assay showed asensitivity of 77% and a specificity of 95%.

A comparison was made of MUC5AC concentration in serum samples fromnormal patients, “early” (stage 1) pancreatic carcinoma and allpancreatic carcinoma samples. The specimens included 13 sera fromhealthy volunteers, 12 sera from stage-1, 13 sera from stage-2 and 25sera from stage-3/4 (advanced) pancreatic carcinoma. A cutoff value of8.8 units/ml (horizontal line) was used, as determined by ROC curvestatistical analysis. The frequency distribution of PAM4 antigenconcentration is shown in FIG. 23, which shows that 92% of “early”stage-1 pancreatic carcinomas were above the cutoff line for diagnosisof pancreatic cancer. An ROC curve for the PAM4 based assay is shown inFIG. 24, which demonstrates a sensitivity of 81.6% and specificity of84.6% for the PAM4 assay in detection of pancreatic cancer.

These results confirm that an enzyme immunoassay based on PAM4 antibodybinding can detect and quantitate PAM4-reactive antigen in the serum ofpancreatic carcinoma patients. The immunoassay demonstrates highspecificity and sensitivity for pancreatic carcinoma. The majority ofpatients with stage 1 disease were detectable using the PAM4immunoassay.

In conclusion, an immunohistology procedure employing PAM4 antibodyidentified approximately 90% of invasive pancreatic carcinoma and itsprecursor lesions, PanIN, IPMN and MCN. A PAM4 based enzyme immunoassayto quantitate MUC5AC in human patient sera showed high sensitivity andspecificity for detection of early pancreatic carcinoma. Due to the highspecificity of PAM4 for pancreatic carcinoma, the mucin biomarker canalso serve as a target for in vivo targeting of imaging and therapeuticagents. ImmunoPET imaging for detection of “early” pancreatic carcinomais of use for the early diagnosis of pancreatic cancer, when it can bemore effectively treated. Use of radioimmunotherapy with a humanizedPAM4 antibody construct, preferably in combination with aradiosensitizing agent, is of use for the treatment of pancreaticcancer.

Example 28 Further Studies of In Vitro Detection of PAM4 Antigen inHuman Serum

In certain embodiments, it is preferred to detect the presence of MUC5ACand/or to diagnose the presence of pancreatic cancer in a subject by invitro analysis of samples that can be obtained by non-invasivetechniques, such as blood, plasma or serum samples. Such ex vivoanalysis may be preferred, for example, in screening procedures wherethere is no a priori reason to believe that an individual has apancreatic tumor in a specific location. The objective of the presentstudy was to develop a reliable, accurate, serum-based assay fordetection of pancreatic cancer at the earliest stages of the disease.

Summary

A PAM4-based immunoassay was used to quantitate antigen in the serum ofhealthy volunteers (N=19), patients with known diagnosis of pancreaticadenocarcinoma (N=68), and patients with a primary diagnosis of chronicpancreatitis (N=29). Sensitivity for the detection of pancreaticadenocarcinoma was 82%, with a false-positive rate of 5% for the healthycontrols. Patients with advanced disease had significantly higherantigen levels than those with early-stage disease (P<0.01), with adiagnostic sensitivity of 91%, 86%, and 62% for stage 3/4 advanceddisease, stage-2, and stage-1, respectively. We also evaluated chronicpancreatitis sera, finding 38% positive for antigen. However, thisobservation was discordant with immunohistochemical findings thatsuggest the PAM4-antigen is not produced by inflamed pancreatic tissue.Furthermore, several of the serum-positive pancreatitis patients, forwhom tissue specimens were available for pathological interpretation,had evidence of neoplastic precursor lesions. Immunohistochemistry ofadditional pancreatitis specimens showed 90% to be PAM4-negative withthe remainder only weakly positive. This suggested that positive levelsof PAM4-antigen within the serum are not derived from inflamedpancreatic tissues, but may be an early indicator of pancreatic cancer.

These results show that the PAM4-serum assay may be used to detectearly-stage pancreatic adenocarcinoma, and that positive serum levels ofPAM4-antigen are not derived from inflamed pancreatic tissues, butrather may provide evidence of subclinical pancreatic neoplasia.

Materials and Methods

Human Specimens

Sera (N=68) were obtained from patients with a confirmed diagnosis ofpancreatic adenocarcinoma being treated at the Johns Hopkins MedicalCenter, Baltimore, Md., and stored frozen <5 yrs. Each of these patientsunderwent surgical resection of the pancreas, providing an opportunityfor accurate diagnosis and staging. For stage-1 disease, no neoplasticcells were observed outside of the pancreas. However, patients withpancreatic adenocarcinoma are likely to have undetected micrometastaticdisease at presentation, including those patients reported with stage-1disease. For this reason, we evaluated follow-up survival data. Allpatients described as having stage-1 disease survived at least 1 year(time to last recorded follow-up visit), with a median survival time of2.70 years (25^(th) percentile=1.32 years) in comparison to the latestSEER data (2002-2006), which reports a 1.42-year median survival forpatients having stage-1 disease treated by surgical resection.

A total of 29 sera from patients with a diagnosis of chronicpancreatitis were obtained from the Johns Hopkins Medical Center andZeptometrix Corp. (Franklin, Mass.). Healthy volunteers (N=19) providedblood for control specimens at the Center for Molecular Medicine andImmunology. All specimens were de-identified, with the only clinicaldata provided to the investigators being the diagnosis, stage ofdisease, follow-up survival time, and size of the primary tumor.

Reagents

A human pancreatic mucin preparation was isolated from CaPan1, a humanpancreatic cancer grown ashttp://jco.ascopubs.org/cgi/content/full/24/2/252-SEC1#SEC1xenografts inathymic nude mice. Briefly, 1 g of tissue was homogenized in 10 mL of0.1 M ammonium bicarbonate containing 0.5 M sodium chloride. The samplewas then centrifuged to obtain a supernatant that was fractionated on aSEPHAROSE®-4B-CL column with the void volume material chromatographed onhydroxyapatite. The unadsorbed fraction was dialyzed extensively againstdeionized water and then lyophilized. A 1 mg/mL solution was prepared in0.01 M sodium phosphate buffer (pH, 7.2) containing 0.15 M sodiumchloride (phosphate-buffered saline [PBS]), and used as the stocksolution for the immunoassay standards. A polyclonal, anti-mucinantiserum was prepared by immunization of rabbits, as describedpreviously (Gold et al., Cancer Res 43:235-38, 1983). An IgG fractionwas purified and assessed for purity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andmolecular-sieve high-performance liquid chromatography. Murine MA5antibody reactive with the MUC1 protein core was obtained fromImmunomedics, Inc. (Morris Plains, N.J.). A non-binding isotype-matchedcontrol antibody, Ag8, was purified from the P3X63-Ag8 murine myeloma.

Sample Preparation

All assays were performed in a blinded fashion. To prepare the specimensfor immunoassay, 300 μL of serum were placed in a 2.0 mL microcentrifugetube and extracted with an equal volume of 1-butanol. The tubes werevortexed vigorously for 2 min at which time 300 μL of chloroform wereadded and the tubes again vortexed for 2 min; this latter step wasincluded in the procedure in order to invert the aqueous and organiclayers. The tubes were then centrifuged in a microfuge at a setting of12,000 rpm for 5 min. The top aqueous layer was removed to a clean tubeand the sample diluted 1:2 in 2.0% (w/v) casein-sodium salt in 0.1 Msodium phosphate buffer, pH 7.2, containing 0.15 M sodium chloride (PBS)for immunoassay.

Enzyme Immunoassay

The immunoassay was performed in a 96-well polyvinyl plate that had beencoated with 100 μL of humanized-PAM4 IgG at 20 μg/mL in PBS withincubation at 4° C. overnight. The wells were then blocked by additionof 200 μL of a 2.0% (w/v) solution of casein in PBS and incubated for1.5 h at 37° C. The blocking solution was removed from the wells and theplate washed 5-times with 250 μL of PBS containing 0.1% (v/v) Tween-20.The standards, or unknown specimens, 100 μL in triplicate, were added tothe appropriate wells and incubated at 37° C. for 1.5 h. The plate wasthen washed 5-times with PBS-Tween-20 as above.

The polyclonal, rabbit anti-mucin antibody, diluted to 5 μg/mL in 1.0%(w/v) casein in PBS containing 50 μg/mL non-specific, human IgG, wasadded to each well and incubated for 1 h at 37° C. The polyclonalantibody was then washed from the wells as above, and peroxidase-labeleddonkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove,Pa.), at a 1:2000 dilution in 1.0% (w/v) casein in PBS, also containing50 μg/mL human IgG, was added to the wells and incubated at 37° C. for 1h. After washing the plate as above, 100 μL of a3,3′,5,5′-tetramethylbenzidine substrate solution were added to thewells and incubated at room temperature for 30 min. The reaction wasstopped by the addition of 50 μL 4.0 N sulfuric acid, and the opticaldensity read at a wavelength of 450 nm using a SPECTRA-MAX® 250spectrophotometer (Molecular Devices, Sunnyvale, Calif.). Because of theconsiderable microheterogeneity of the PAM4 antigen, we chose to reportour results in arbitrary units/mL, based on an initial referencestandard purified from xenografted CaPan-1 human pancreatic tumor.

Immunohistochemistry

Paraffin-embedded specimens obtained from the Cooperative Human TissueNetwork were cut to 4 micron sections on superfrost plus adhesive slides(Thermo Scientific, Waltham, Mass.). Tissue sections were then heated to95° C. for 20 min in a pH 9.0 Tris buffer, Target Retrieval Solution(Dako, Carpinteria, Calif.), allowed to cool to room temperature, andthen quenched with 3% H₂O₂ for 15 min at room temperature. Primaryantibodies were then used at 10 μg/mL with an ABC VECTASTAIN® kit(Vector Laboratories, Burlingame, Calif.) for labeling the tissues. Theslides were scored independently by two pathologists using a paradigmconsistent with that reported for earlier studies on biomarkers inpancreatic adenocarcinoma (Gold et al., 2007, Clin Cancer Res13:7380-87): 0-negative, <1% of the tissue was labeled; 1-a weak, focallabeling of between 1%-25% of the tissue; 2-a strong, focal labeling ofbetween 1%-25% of the tissue; 3-a weak, diffuse labeling >25% of thetissue; 4-a strong, diffuse labeling >25% of the tissue. Only theappropriate tissue components (e.g., adenocarcinoma cells, normal ducts,etc.) were considered for assessment.

Statistical Analyses

Standard curves were generated from the immunoassay data, withregression analyses performed to interpolate concentrations of theunknown samples (Prism 4.0 software, GraphPad, La Jolla, Calif.).Receiver operating characteristic (ROC) curves were generated by use ofthe Med-Calc statistical software package (version 7.5) (Med-Calc,Mariakerke Belgium). Student's t-test was used to compare variables inany two groups. The Cochran-Armitage test was used to detect a trendbetween detection rates and stage of disease.

Results

Accuracy and Precision of the Immunoassay

A set of control standards with nominal concentrations of 15.60, 6.20,2.50, and 1.00 units/mL was evaluated on several nonconsecutive days(N=7) for determination of accuracy and precision. Curve fitting for thestandards generally gave resultant goodness of fit values for r²>0.990.Accuracy was calculated to be within 8% of the nominal value for thefirst three concentrations, but fell to approximately 22% for the 1.00units/mL standard. Linear regression of nominal vs. measured units/mL inthis series of controls gave a trend-line with a slope of 0.965 and yintercept of 0.174 (r²=0.999), where a slope of 1.00 with a y interceptof 0.00 would constitute 100% accuracy (FIG. 25). An average absolutedifference between nominal and recovered mass equal to 0.190±0.173units/mL for the two lowest concentration standards suggested a minimumabsolute error of approximately 0.2 units/mL for the EIA. Values for thecoefficient of variation (CV) were 6.40%, 4.85%, 12.0%, and 66.4%,respectively, for the 4 control standards. Taken together, the datasuggest that the PAM4-immunoassay provides levels of accuracy andreproducibility that are within the guidelines suggested for animmunoassay measurement of an analyte; accuracy and precision werewithin 15% for concentrations above the cutoff value (2.40 units/mL),and within 20% at the cutoff value. To further test this, we examined 3sera, two of which were from healthy controls, on 3 separate days. Thetwo healthy controls gave average results of 0.27±0.06 and 0.30±0.27units/mL, each of which was close to the minimum absolute error for theEIA with consequent high CV of 21.65% and 88.19%, respectively. Theother patient serum gave an average of 19.45±2.51 units/mL with a CV of12.9%.

Quantitation of Antigen in Patient Sera

In a preliminary study reported in the Example above, the PAM4serum-based immunoassay had an apparent sensitivity of 77% and aspecificity of 94% for pancreatic carcinoma. It should be noted that theoverwhelming majority of cancer specimens of pancreatic andnon-pancreatic origin had been obtained from patients enrolled inIRB-approved clinical trials conducted by the Garden State Cancer Centerand stored frozen at −80° C. for more than 10 yrs. However, thespecimens of pancreatitis had been stored frozen for a considerablyshorter time. We evaluated a new group of 24 sera from patientsdiagnosed with pancreatic adenocarcinoma. Only two of the sera hadlevels of PAM4-reactive antigen considered to be positive. Therefore, weconsidered and evaluated reasons why the immunoassay had not performedas expected, including the quality of the immunoassay reagents, thepossibility that the antigen was being degraded and/or removed from theserum, its presence in the form of immune complexes, or being bound by ablocking substance. We discovered that there is a substance in freshhuman serum and/or specimens stored frozen for short periods of time (<5yrs) that apparently binds to the PAM4-reactive epitope and blocks itsbinding to PAM4 antibody, thus preventing detection by immunoassay.Percent recovery of antigen from fresh normal human serum (N=2) spikedwith PAM4-antigen at concentrations from 5-20 units/mL was on the orderof 33% or less.

In a series of reports, Slomiany and co-workers disclosed that gastricmucin had covalenty bound and/or associated lipids and fatty-acids(Slomiany et al., 1984, Arch Biochem Biophys 229:560-67; Slomiany etal., 1986, Biochem Biophys Res Commun 141:387-93; Zalesna et al., 1989,Biochem Int 18:775-84), and that these lipids and fatty acids hadspecific effects upon the physicochemical properties of the mucin.Furthermore, it was reported that fatty-acid synthetase levels andactivity are significantly elevated in pancreatic adenocarcinoma, as isalso the case for other forms of cancer and other pathologic conditions(Walter et al., 2009, Cancer Epidemiol Biomarkers Prev 19:2380-85).Because the blocking substance might be lipid in nature, we performedorganic extraction of sera from the group of 24 pancreaticadenocarcinoma patients that had been stored frozen for <5 years. As wasnoted above, without prior extraction, only 2 of the 24 specimens (8.3%)had levels of PAM4-antigen that were considered positive, whereas afterorganic extraction, 22 of the 24 specimens (92%) had positive levels ofthe PAM4-antigen.

We were also able to re-evaluate, from the study reported in the Exampleabove, 10 pancreatic adenocarcinoma patient sera that had been storedfrozen for >15 years to confirm the prior results. With or withoutextraction, all 10 specimens had levels of antigen that were consideredto be positive. Regression analysis to compare paired results fromextracted and non-extracted sera gave a trendline with slope of 1.10(r²=0.94), demonstrating that with or without extraction of theselong-term frozen sera, the results were similar. It is considered thatlong-term storage of the specimens resulted in degradation of theinhibiting substance or decreased binding to and unmasking of theepitope. All further testing of sera was performed with organicextraction of specimens prior to immunoassay.

Specimens evaluated for PAM4-reactive antigen included 68 patients withconfirmed pancreatic adenocarcinoma divided by stage: 21 from stage-1;14 from stage-2; and 33 from stages-3 and -4 (advanced). In addition, 19sera collected from healthy adult volunteers and 29 patients diagnosedwith chronic pancreatitis were included as control groups. The maximumconcentration shown in the dot-plot (FIG. 26) was 80 units/mL, becausethere were insufficient volumes of sera to perform additional dilutionstudies. Although a cutoff value of 10.2 units/mL was reported in theExample above, because of the use of an organic extraction procedure, aswell as differences in the EIA protocol (reagent concentrations,inclusion of human IgG in buffers), we chose to treat the current dataset independently of prior results. A positive cutoff value of 2.4units/mL was calculated by ROC curve statistics (FIG. 27) for thecomparison of all pancreatic adenocarcinoma specimens versus healthyadults. The overall sensitivity for detection of pancreaticadenocarcinoma was 82%, with an area under the curve of 0.92±0.03 (95%CI=0.84-0.97). At this level of sensitivity, a false-positive rate of 5%was observed for the healthy control group, the single positive casehaving 3.65 units/mL of circulating antigen, just above the cutoffvalue. Insufficient volumes of sera precluded CA19-9 immunoassays forcomparison to the PAM4-immunoassay results.

As shown in Table 20, sensitivity for detection of early, stage-1pancreatic adenocarcinoma was relatively high, with 13 of 21 (62%)specimens above the cutoff value. As expected, this detection rate waslower than that observed for the stage-2 (86%) and advanced stage-3 and-4 (91%) patient groups. A statistically significant trend (P<0.01) wasnoted for detection rate vs. stage of disease. We considered that thiswas most likely due to tumor size or burden. The average tumor sizes forstage-1, stage-2, and stage-3/4 groups were 2.14±1.02 cm³, 3.36±1.18cm³, and 3.45±1.06 cm³, respectively. While there was no statisticallysignificant difference in tumor size between the stage-2 and -3/4 groups(P>0.41), a statistically significant difference was observed for eachof these two groups when compared to stage-1 tumor size (P<0.004 orbetter). However, it should be noted that individual tumor size did notcorrelate with antigen concentration in the serum (r²=0.0065).

Specimens reported as Stage-1 could be divided into stage-1A (N=13) andstage-1B (N=8) subgroups based on tumor size, with detection rates of54% and 75%, respectively; however, caution is emphasized since thenumber of patients in each subgroup is small. The average tumor size forstage-1A was 1.41±0.58 cm³ (range: 0.4 cm³-2.0 cm³) and for stage-1B was3.15±0.44 cm³ (range: 2.5 cm³-4 cm³); P<0.001 for comparison of the twogroups. While, on the whole, tumor sizes were smaller in stage-1Adisease than in stage-1B, there was no apparent statistical correlationbetween individual tumor size and concentration of the PAM4-antigen inthe blood (r²=0.03). Furthermore, it is important to note that of the 13stage-1A specimens, 4 of the 7 positive cases had PAM4-antigen levelsconsiderably higher than the cutoff value, with a range of 17.65-32.65units/mL.

TABLE 20 PAM4-reactive antigen in the sera of patients Median T-test N(units/mL) True-Positive (P value)^(a) Total PC 68 9.85 81% <0.001Stage-1 21 4.53 62% <0.002 Stage-1A 13 3.96 54% <0.02 Stage-1B 8 6.0575% <0.02 Stage-2 14 10.39 86% <0.005 Stage-3/4 33 13.37 91% <0.001Chronic Pancreatitis 29 1.28 (38% FP) Healthy Volunteers 19 1.18  (5%FP) ^(a)All comparisons are to healthy volunteers

We also evaluated a set of 29 patient sera with the primary diagnosis ofchronic pancreatitis. At the 2.4 units/mL cutoff established by ROCevaluation of normal and pancreatic adenocarcinoma patients, 11pancreatitis patients (38%) were positive. ROC curve analysis ofpancreatitis sera compared directly to the pancreatic adenocarcinomaspecimens gave an area under the curve of 0.77±0.05 (95% CI=0.68-0.85).The median value for the pancreatitis group was 1.28 units/mL,comparable to the healthy volunteer group (1.18 units/mL), butconsiderably lower (3.5-fold) than the stage-1 pancreatic adenocarcinomagroup (4.53 units/mL). It should be noted that our earlier results forpancreatitis specimens suggested a considerably lower false-positiverate, only 5%. However, those pancreatitis specimens were stored frozenfor less than 5 years, and were not organic phase extracted prior toanalysis.

Biopsy and/or surgical specimens were available from 14 of the chronicpancreatitis specimens, 6 of which were from patients who wereconsidered positive for circulating MUC5AC. In 3 of these 6 positivecases, precursor lesions were identified within the tissue sections. Itwas then considered whether the positive serum test was due topancreatitis or the presence of neoplastic precursor lesions. Weperformed immunohistochemistry on an additional 30 biopsy specimens frompatients diagnosed with pancreatitis. Of the 30 specimens, one frankinvasive pancreatic adenocarcinoma and one large PanIN-2-3 lesion wereidentified (in separate specimens) by use of PAM4 staining, whilesurrounding acinar-ductal metaplasia (ADM) and normal tissues werenegative (data not shown). Of the remaining 28 specimens, 19 hadsufficient parenchyma to be evaluated, 16 of which had evidence of ADM.PAM4 was negative in all but two of these cases, and in each of thesegave only a very focal, weak labeling of ADM within the specimens (datanot shown).

Validation Studies—

We have begun putting together a panel of well-annotated serum specimensfrom patients with known diagnoses. A first set of patient sera (N˜450)including healthy individuals and patients having invasive pancreaticcancers (carcinoma, neuroendocrine, and other forms), benign disease ofthe pancreas (adenomatous lesions, pancreatitis, etc.) andnon-pancreatic cancers and benign disease (biliary, duodenal, ampullarycarcinomas, cholecystitis, gastritis, etc.) is being evaluated (blindstudy) to both confirm and extend the prior results on PAM4 specificityin a much larger group of patients. Table 21 presents an interimanalysis based upon studies completed to date; overall, the data areremarkably similar to our earlier data. Employing a cutoff valuedetermined by ROC analysis of PC vs Healthy Adults, the overallsensitivity for detection of pancreatic carcinoma was 80% at aspecificity of 96%. Only 2 of 16 neuroendocrine tumors were positive,just over the cutoff value.

To date, 14 of 53 (26%) patients with primary diagnosis of pancreatitishave been identified as PAM4 positive, lower than that reported in ourrecent publication. We are now attempting to correlate clinical datawith results in this pancreatitis group, as well as provide for clinicaland laboratory follow-up of these patients. Only 2 of 11 patients withbenign adenomatous lesions (both cystadenoma) were considered positive.One other cystadenoma had PAM4-antigen levels greater than 200 units/mL.The pathology report describes the biopsy as “very suspicious forcancer”.

TABLE 21 PAM4-reactive antigen in the sera of patients PositiveROC-AUC^(b) N Median^(a) Mean ± SD^(a) (%) (95% CI) P value^(c)Pancreatic 145 7.84 35.61 ± 64.58 80 (comparisons Carcinoma are to PC)Neuroendocrine 16 0.00 1.73 ± 4.91 12.5 Healthy 27 0.40 0.54 ± 0.53 3.70.90 ± 0.02 <0.0001 (0.85-0.94) Pancreatitis 53 0.37 1.56 ± 3.35 26 0.85± 0.28 <0.0001 (0.79-0.90) Pancreatic 11 0.00 0.64 ± 0.78 18 0.90 ± 0.03<0.0001 Adenoma (0.84-0.94) ^(a)Values for Median and Mean ± SD are inUnits/mL ^(b)Receiver Operating Characteristic Curves (ROC); Area Underthe Curve (AUC) with values for 95% Confidence Intervals presented.

Discussion

Studies reported in the Example above that employed both immunohistologyof tissue specimens and EIA of circulating antigen demonstrated that thePAM4-reactive epitope is a biomarker for invasive pancreaticadenocarcinoma and is expressed at the earliest stages of pancreaticneoplasia (i.e., PanIN-1). It was not detectable within normalpancreatic tissues (ducts, acinar and islet cells), nor the majority ofnon-pancreatic cancers examined (breast, lung, gastric, and others).Thus, an elevation of the PAM4-epitope concentration in the serumprovided a high positive likelihood ratio of 16.8 for pancreaticadenocarcinoma. Missing from the prior study was clinical informationregarding the stage of disease. Consequently, we could not evaluate thevalue of the immunoassay for detection of potentially curable earlydisease until now.

We report herein that PAM4-based EIA using serum samples can detectpatients having early-stage pancreatic adenocarcinoma, and can provideaccurate discrimination from disease-free individuals. The assay'ssensitivity for detection of early pancreatic adenocarcinoma was 62% forpatients with stage-1 and 86% for patients with stage-2 disease andserum levels generally increased with advancing stage of disease. A highpercentage of patients with stage-1 and -2 disease are clinicallyasymptomatic. We conclude that detection of tumor growth at these earlystages using a PAM4 serum assay could provide improved prospects forsurvival.

The cancer patients in this study all underwent surgical resection,providing an opportunity to accurately stage each patient. However, manypatients with pancreatic cancer are suspected of having micrometastaticdisease at presentation, even if they do not havehistologically-apparent regional lymph node involvement. This highlightsa general problem in the study of early detection, particularly with alow-incidence disease such as pancreatic adenocarcinoma. The accrual ofspecimens that are well-defined is problematic. Further complicating theissue is that many of these pancreatic cancers occur in the presence ofchronic pancreatitis, cholecystitis, and neoplastic precursor lesions,amongst other conditions.

Of 29 sera with a primary diagnosis of chronic pancreatitis, 38% wereidentified as positive for PAM4-antigen. However, several of theseserum-positive patients, for whom tissue specimens for pathologicalinterpretation were available, had evidence of neoplastic precursorlesions. Furthermore, a discrepancy was observed in the comparison oftissue reactivity by immunohistology and serum levels of antigen byimmunoassay. By immunohistochemistry, only 10% of the evaluablespecimens showed evidence of PAM4 staining within the ADM, although thiswas at considerably lower intensity than observed for the overwhelmingmajority of pancreatic adenocarcinoma specimens (Gold et al., 2007, ClinCancer Res 13:7380-87). Therefore, the results suggest that positivelevels of PAM4-antigen within the serum may not be derived from inflamedpancreatic tissues, but rather could provide evidence of subclinicalpancreatic neoplasia, such as PanIN lesions, and that, at the veryleast, positive results provide the rationale for clinical follow-up ofthese patients.

Findings from genetically-engineered animal models of pancreaticadenocarcinoma suggest that human pancreatic neoplasia may arise beforethe PanIN-1 lesion (Leach, 2004, Cancer Cell 5:7-11). ADM was theearliest change observed in the mutant KRAS targeted model described byZhu et al. (2007, Am J Pathol 171:263-73). On the other hand, Shi et al.(2009, Mol Cancer Res 7:230-36) reported that although KRAS genemutations can occur within ADM, they occur predominantly within ADM thatare associated with PanIN lesions. The authors suggest this may occur byretrograde extension of the PanIN to the surrounding ADM. As yet, thereis no conclusive evidence that ADM progress to PanIN. The fact that PAM4is reactive with ADM in two patients with pancreatitis is of interest.

At the present time, screening the general population for pancreaticcancer is not considered medically or economically worthwhile, becausethe disease is simply too infrequent. However, there is considerableinterest in screening patients predicted to have an increased risk ofdeveloping pancreatic adenocarcinoma. Several studies have demonstratedthat screening individuals with strong family histories of pancreaticcancer can identify precursor neoplasms of the pancreas that areamenable to surgical resection (Canto et al., 2006, Clin GastroenterolHepatol 4:766-81; Canto, 2005, Clin Gastroenterol Hepatol 3:S46-58;Brentnall et al., 1999, Ann Intern Med 131:247-55). For example,relatives of pancreatic cancer patients have a significantly higher riskof developing pancreatic cancer than the general population (Shi et al.,2009, Arch Pathol Lab Med 133:365-74). A small percentage of patientswith familial pancreatic cancer harbor mutations of PALB2 (partner andlocalizer of BRCA2), a susceptibility gene for pancreatic cancer(Tischkowitz et al., 2009, Gastroenterology 137:1183-86). Similarly,patients with long-standing chronic pancreatitis are at increased riskof developing pancreatic cancer, and the risk is over 30%, amongpatients with early-onset (teenage) hereditary pancreatitis (Lowenfelset al., 1993, New Eng J Med 328:1433-37; Lowenfels et al., 1997, J NatlCancer Inst 89:442-46). A 20- to 34-fold higher risk has been observedin individuals with familial atypical multiple mole (FAMMM) syndrome(Rutter et al., 2004, Cancer 101:2809-16). Also, several studies haveshown a significantly increased risk of developing pancreatic cancer indiabetic individuals who meet certain criteria (Pannala et al., 2009,Lancet Oncol 10:88-95). Longitudinal surveillance of these patients byuse of the PAM4-immunoassay may provide for early detection ofneoplasia. A second potential use of the immunoassay could be as a meansto detect recurrence of disease post-therapy, and in particular,following surgical resection for those patients where the tumor issupposedly confined to the pancreas.

The relatively high specificity of the PAM4 antibody provides a means totarget both imaging and therapeutic agents with high tumor uptake andhigh tumor/nontumor ratios. We have demonstrated PAM4's potential asboth a directly-radiolabeled or bispecific, pretargeting reagent fornuclear imaging and radioimmunotherapy of pancreatic cancer. Also,initial results of a clinical phase 1b trial to evaluate a fractionateddosing of ⁹⁰Y-PAM4 whole IgG (clivatuzumab tetraxetan), in combinationwith a radiosensitizing regimen of gemcitabine, were reported recently(Pennington et al., 2009, J Clin Oncol 27:15s, abstract 4620). Of 22patients with stage-3/4 disease (mostly stage-4), 68% showed evidence ofdisease control, with 23% of patients having partial responses based onRECIST criteria. Thus, positive results by the PAM4-based immunoassayprovides a rationale to pursue PAM4-targeted imaging and therapy, thusproviding a personalized therapy.

The PAM4-based immunoassay can identify the majority of pancreaticadenocarcinoma patients of all stages. Although a direct comparison withCA19.9 was not possible in the current study, a prior comparison of thetwo biomarkers in a limited set of pancreatic adenocarcinoma sera (N=41)demonstrated a statistically significant difference (P<0.01) withPAM4-antigen levels positive in 71% of patient specimens andCA19.9-antigen levels positive in 59% of specimens. In general, it isthought that CA19.9 lacks the sensitivity and specificity to provide forearly detection and/or diagnosis of pancreatic adenocarcinoma. However,the assay does have its use for management with continued elevation inCA19.9 serum levels post treatment indicative of a poor prognosis.Similarly, we recently reported in abstract form (Pennington et al.,2009, J Clin Oncol 27:15s, abstract 4620), the use of circulatingPAM4-antigen levels for prediction of anti-tumor response.

These results show that the conditions under which specimens are stored(e.g., the length of time they are kept frozen) can have significanteffects upon accessibility of the epitope under study. For thePAM4-based immunoassay, a fatty acid or lipid substance may be able tobind the specific epitope and interfere with the immunoassay. However,it is also possible this material was a low-molecular weight peptide orother substance soluble in organic solvents. The ability to remove thissubstance by organic extraction of the serum makes the PAM4-immunoassayreproducible. In addition, the question is raised as to the biologicalsignificance of the circulating inhibitor:MUC5AC interaction. However,when using the PAM4 antibody as an in vivo targeting agent (e.g.,radioimmunotherapy), the presence of circulating PAM4-antigen is not afactor, since targeting of radiolabeled-PAM4 to sites of tumor growthhas been observed in the majority of patients evaluated to date. Thus,it appears that the PAM4-antigen within tumor is free of the blockingsubstance.

Example 29 Phase IB/II Study of ⁹⁰Y-Labeled hPAM4 Antibody andGemcitabine in Advanced Pancreatic Cancer

A phase IB/II study of ⁹⁰Y-labeled hPAM4 antibody (clivatuzumabtetraxetan) in advanced pancreatic cancer patients was performed. Atotal of 100 patients with previously untreated Stage III or IVpancreatic cancer were enrolled into this open-label trial to receivegemcitabine once-weekly×4 with ⁹⁰Y-clivatuzumab tetraxetan on weeks 2, 3and 4 (therapy cycle). The therapy cycle could be repeated until diseaseprogression or until the patient displayed unacceptable toxicity. Tenpatients withdrew early, while 90 patients, of whom 82 had the Stage IV(metastatic) disease, received 1-4 therapy cycles. Tumor responses wereassessed by CT, FDG/PET and serum CA19.9 after each cycle (initiallyevery 4 wks).

In Part I of this study, 38 patients were treated with ⁹⁰Y-clivatuzumabtetraxetan at 6.5, 9, 12 or 15 mCi/m²×3, and a low, fixed gemcitabinedose of 200 mg/m²×4 for radiosensitization. Thirteen patients wereretreated with the same cycle 1-3 times. The overall disease controlrate, which included complete response (CR), partial response (PR) andstable disease (SD), by CT-based RECIST criteria, was 58%, including 6patients (16%) with PR and 16 patients (42%) with SD as best response.

The median overall survival (OS) for the 38 treated patients was 7.7months, which compares favorably with other regimens for advancedpancreatic cancer. At the higher therapy doses (12 and 15 mCi/m² of⁹⁰Y-clivatuzumab tetraxetan×3), a median OS of 8.0 months was noted. Forthe 13 patients who received repeated cycles of the combination therapy,median OS improved to 11.8 months. Extended survival of up to 14.8months post therapy onset has been observed, with 8 patients achieving asurvival >6 months (3 patients >1 yr). Anecdotal reports indicateperformance status and pain level improved with therapy.

Fifty-two patients who were treated in Part II of this study received 3weekly ⁹⁰Y doses of 12 mCi/m² and gemcitabine doses of 200, 600 or 1000mg/m²×4, with 14 patients receiving repeated therapy cycles at the samegemcitabine dose but ⁹⁰Y doses of 6.5, 9 or 12 mCi/m². Results wereavailable from 47 of the 52 patients. The disease control rate for the200 mg/m² group was 72%, with 19% PR and 53% SD. For the 600 and 1000mg/m² groups, the disease control rates were 63% (0% PR) and 68% (18%PR), respectively. Higher gemcitabine doses appeared to offer noadvantage in treatment response over the lowest dose of 200 mg/m². Atthe time of reporting, survival data were not available for this groupof patients. Treatments were well tolerated with no infusion reactionsto radiolabeled clivatuzumab and few non-hematologic side effects.Hematologic suppression was transient after cycles 1 and 2.

These results showed that repeated cycles of fractionated doses ofclivatuzumab tetraxetan, labeled with yttrium-90 (⁹⁰Y) and given incombination with gemcitabine, demonstrated therapeutic activity inpatients with advanced, inoperable, pancreatic cancer. Therapy withrepeated cycles of clivatuzumab tetraxetan plus low-dose gemcitabineimproved overall survival over single-cycle therapy in patients withlocally advanced or metastatic pancreatic cancer.

Example 30 Detection of Early-Stage Pancreatic Ductal Adenocarcinoma(PDAC): Sensitivity, Specificity, and Discriminatory Properties ofSerum-Based PAM4-Immunoassay

As disclosed in Example 28, a serum-based enzyme immunoassay employingthe PAM4 antibody was able to correctly identify 81% of patients withknown PDAC and this assay had promising sensitivity for detectingearly-stage disease. These findings have been extended in a much largerpatient population that included over 600 sera from both malignant andbenign diseases of the pancreas and surrounding tissues. In a blindedanalysis, sera from patients with confirmed PDAC (N=298), other cancers(N=99), benign disease of the pancreas (N=126), and healthy adults(N=79) were evaluated by enzyme immunoassay for concentration ofPAM4-antigen levels.

Overall sensitivity for detection of PDAC was 76%, with 64% of stage-1patients testing positive and a higher sensitivity (85%) for advanceddisease. For the most part, sera from patients with neuroendocrinetumors of the pancreas or cancers of other origin (squamous, GIST, etc.)did not have elevated levels of the PAM4-antigen. Approximately half ofthe patients with ampullary (48%) and extrahepatic biliary (50%)adenocarcinomas had positive levels of circulating PAM4-antigen. Of 126patients diagnosed with benign conditions of the pancreas, only 24 (19%)were positive and, in particular, 18 of 80 (23%) patients with chronicpancreatitis (CP) were positive. ROC curve analysis demonstrated astatistically significant difference between the PDAC and CP groups(P<0.0001), with an area under the curve of 0.84±0.02 (95% CI:0.79-0.89). The positive- and negative-likelihood ratios fordifferentiating PDAC from benign conditions of the pancreas were 4.00and 0.30, respectively.

In conclusion, the PAM4-immunoassay detected nearly two-thirds ofstage-1 PDAC patients, and did so with high discriminatory power withrespect to benign pancreatic disease. The results provide a rationalefor longitudinal surveillance of patients considered at high-risk forPDAC (e.g., familial pancreatic cancer, new-onset diabetes, etc.) withthe PAM4 assay.

Example 31 PAM4-Based Assay Differentiates Pancreatic DuctalAdenocarcinoma (PDAC) from Chronic Pancreatitis and Benign NonmucinousPancreatic Cysts

We examined the expression of PAM4-reactive MUC5AC in chronicpancreatitis and benign non-mucinous cystic lesions of the pancreas. Atissue microarray of PDAC (N=14), as well as surgical specimens fromchronic pancreatitis (N=32) and benign non-mucinous cystic lesions ofthe pancreas (N=19), were assessed by immunohistochemistry forexpression of the PAM4-reactive MUC5AC, as well as MUC1 (mAb-MA5), MUC4(mAb-8G7), and CEACAM6 (mAb-MN-15).

PAM4-reactive MUC5AC, MUC1, MUC4 and CEACAM6 were expressed in 79%(11/14), 100% (14/14), 86% (12/14) and 100% (14/14) of invasivepancreatic adenocarcinoma. PAM4 only weakly labeled 6% (1/19) of benignnon-mucinous cystic lesions, 1 of 15 serous cystadenomas (SCAs) and 0 of4 cysts with squamous epithelial lining (2 lymphoepithelial cysts, and 2retention cysts with squamous metaplasia). However, the expression ofMUC1, MUC4 and CEACAM6 was detected in 53% (8/15), 0% (0/15) and 13%(2/15) of SCAs, and in 4, 3 and 3 of the 4 cysts with squamousepithelial lining, respectively. PAM4 labeled 19% (6/32) of chronicpancreatitis specimens; however, this PAM4 reactivity was restricted tothe PanIN precursor lesions associated with chronic pancreatitis.Inflamed tissue was negative. The expression of MUC1, MUC4 and CEACAM6was detected in 90% (27/30), 78% (25/32), and 97% (31/32) of chronicpancreatitis. In all of the positively-labeled specimens, the reactivitywas present in non-neoplastic inflamed pancreatic tissue in addition toPanIN.

In conclusion, the expression of PAM4 was detected in only 6% of benignnon-mucinous cystic lesions and in the precursor lesions associated withchronic pancreatitis. These results suggest that PAM4, in contrast toMUC1, MUC4, and CEACAM6, may be useful to differentiate benignnon-mucinous cystic lesions of the pancreas and chronic pancreatitisfrom PDAC.

Example 32 Combination of the PAM4 and CA19-9 Biomarkers for ImprovedDetection of Pancreatic Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal,due mainly to the inability to detect early-stage disease. Thus,identification of biomarkers that can identify patients with early-stagePDAC may improve overall survival. In a blinded study, PAM4 and CA19-9immunoassays were performed on sera from 480 patients, including thosewith confirmed PDAC (N=234), other cancers (N=84), benign diseases ofthe pancreas (N=89), and healthy adults (N=50).

Overall sensitivity for PDAC was similar, 74% and 77% for PAM4 andCA19-9, respectively. Sensitivity for detection of early, stage-1disease (N=26), although somewhat higher for the PAM4-antigen, was alsostatistically similar, 65% and 58% for PAM4 and CA19-9, respectively(P=0.5775). However, specificity was significantly lower for CA19-9,particularly with respect to chronic pancreatitis (CP): 68% vs. 86% forthe PAM4 assay (P=0.014). Furthermore, CA19-9 results showedconsiderably higher detection rates for non-PDAC neoplasia, includingpatients with other cancers that metastasized to the pancreas. Thus,positive likelihood ratios (+LR) were lower for CA19-9 (+LR=2.41) thanfor the PAM4 assay (+LR=5.29).

PAM4 and CA19-9 antigen levels in PDAC were independent of each other(r²=0.003, P=0.410); however, the positive and negative interpretationswere concordant in 68% of the cases. Thus, a combined biomarker analysisimproved the overall PDAC detection rate (84%), without a significantdecrease in specificity (83%). Comparison of the ROC curves for PDAC vs.CP and PDAC vs. benign disease demonstrated a statistically significantimprovement for the combined immunoassay, as compared to either assayalone (P<0.0001 in both comparisons), to detect and discriminate PDACfrom benign disease.

While the PAM4-immunoassay provided high sensitivity and specificity fordetection and diagnosis of PDAC, inclusion of the CA19-9 biomarkersignificantly enhanced positive identification of PDAC patients, from74% to 84%.

Example 33 Use of PAM4-Immunoassay as a Correlate of Tumor Response

We investigated whether specific trends in PAM4-reactive MUC5ACconcentrations (within the individual patient) can be used as anindicator of tumor response after therapy. Several patients from a⁹⁰Y-hPAM4 phase-1b/II clinical trial now in progress were evaluated.When patients were evaluated 4 weeks after treatment had ended (atreatment cycle is 4 weeks), a decrease in serum antigen levels of >40%was suggestive of a response. All of the patients who had progressivedisease had levels of PAM4 antigen that continued to rise. Trends arepresented for two patients in FIG. 28A and FIG. 28B. In both cases,trends in the level of circulating MUC5AC were concordant with the trendin tumor volume as determined by CT. These results suggest that serumPAM4 levels are of use to monitor responsiveness to anti-cancertreatments for pancreatic cancer.

Example 34 Identification of Target Antigen for PAM4 Antibody

We performed a set of blocking and capture/probe paired enzymeimmunoassays to evaluate the relationships between the PAM4 antibody andantibodies reactive with MUC1 (MA5, KC4, HMFG1, SM3, H23), MUC2 (G9),MUC4 (8G7) and MUC5AC (45M1). A mucin standard derived from the CaPan1human tumor xenograft was shown to contain the reactive mucin speciesfor all of these antibodies except those reactive with G9 (MUC2). Of allMAbs examined, only 1 (45M1) reported to be reactive with MUC5ACprovided a positive reaction in sandwich EIA when PAM4 was used as thecapture reagent. The 45M1 antibody is reactive with a much lowerpercentage of pancreatic carcinomas than PAM4 (by IHC on TMA) and socannot be used as a single probe for the serum-based PAM4-immunoassay.

As described above, we performed a peptide-phage-display study byconsecutive biopanning with the murine and humanized versions ofPAM4-IgG. A consensus sequence (12mer—WTWNITKAYPLP (SEQ ID NO: 7)) wasgenerated which when input into a BLAST protein search with querycoverage set at 100%, identified MUC5AC and MUC16 with 7 of 12 and 5 of12 identical amino acids within the 12mer sequence, respectively.

Studies were performed using mass spectrometry to identifyPAM4-immunoprecipitated antigens from credentialed cyst fluids (thesefluids were previously analyzed by mass spectrometry to identifyspecific MUCs present in the mixtures). By PAGE analyses of thePAM4-immunoprecipitated materials from 3 individual cyst fluidspecimens, only two identical bands were present in each specimen (notshown). Both of these bands contained MUC5AC as the major mucin species.

We have investigated the nature of the substance within human blood thatbinds to the PAM4-epitope, which necessitates organic extraction priorto immunoassay. As discussed above, Slomiany and co-workers haveobserved that gastric mucin had covalently bound and/or associatedlipids and fatty-acids. Further, fatty-acid synthetase levels andactivity are significantly elevated in pancreatic adenocarcinoma, as isalso the case for other forms of cancer and other pathologic conditions.Speculating that the blocking substance might be lipid in nature, weperformed an EIA (FIG. 29) in the presence and absence of 100 μMpalmitic acid and observed a statistically significant 69% reduction inreactivity at an OD450 equivalent of 1.0 (P<0.0001). It is noted thatthe normal adult serum level of palmitic acid is in the range of 1,480to 3,730 μM, considerably higher than the concentration that was used inthis EIA experiment.

Example 35 PAM4 Differentiates Between Pancreatic Ductal Adenocarcinoma(PDAC) and Chronic Pancreatitis (CP)

Current practice guidelines suggest that patients who present with signsand/or symptoms suspicious of pancreatic cancer undergo a pancreaticprotocol CT imaging study for detection of tumor mass within thepancreas. Follow-up imaging by endoscopic technologies (e.g., EUS, ERCP)can provide high sensitivity for detection of disease, and when combinedwith fine-needle aspiration/biopsy, can provide good diagnosticaccuracy. However, the majority of these procedures have been performedon patients with advanced disease; that is, tumors greater than 2 cm.Detection of early pancreatic cancer is still problematic, especiallywhen occurring in a background of pancreatitis. Thus, the currentreality is that only 7% of all cases detected are early disease. With noeffective treatment for advanced PC, the prognosis for these patients isdismal.

Biomarkers that can reliably distinguish between cancer and benignconditions, and/or provide means to prioritize patients for follow-upevaluation, would be of significant clinical value, especially if thebiomarker is capable of detecting early disease. We have developedmonoclonal antibody PAM4 that demonstrates a high degree of specificityfor pancreatic ductal adenocarcinoma (PDAC).

MAbs having defined reactivity with several mucin species, includingMUC1, MUC2, MUC3, MUC4, MUC5AC, etc., were evaluated for signal responsein a heterologous PAM4-capture sandwich EIA. The only MAbs able toprovide signal response (45M1, 2-11M1) are known to react with specificdomains of the MUC5AC mucin. Further, three additional anti-MUC5AC MAbs(21M1, 62M1, and 463M1) were each able to inhibit the interactionbetween PAM4 and its mucin antigen. These data suggest MUC5AC as anantigen to which PAM4 is reactive. PAM4, unlike other anti-MUC5AC MAbs(45M1, 2-11M1, CLH2, and others), demonstrates greater specificity forPDAC than cancers originating from other organs, and may serve as auseful biomarker for PDAC, as well as a target for antibody-directedimaging and therapy.

TABLE 22 PAM4-Antigen In the Serum of Patients with Known Disease Numberof Percent of Median Positive Positive Pancreatic Cancer N (units/mL)Cases Cases Ductal Adenocarcinoma 298 10.40 225 76 Neuroendocrine 200.08 2 10 Other Morphology 7 0.51 1 14 Non-PC, Mets to the Pancreas 110.00 2 18 Ampullary Adenocarcinoma 21 1.52 10 48 Biliary Adenocarcinoma26 4.41 13 50 Cholangiocarcinoma 7 1.07 2 29 Duodenal Adenocarcinoma 72.80 4 57 All Biliary and Periampullary 61 1.78 29 48 Colon Carcinoma 320.15 5 16 Chronic Pancreatitis (CP) 80 0.41 18 23 Benign Cystadenoma 150.18 1 7 Benign - Other 25 0.20 5 20 All Benign Disease 120 0.26 24 20Healthy Volunteers 79 0.27 3 4 All groups are statistically differentfrom the pancreatic adenocarcinoma group with P values equal to orbetter than 0.0001; Mann-Whitney nonparametric test. Gold DV, Gaedcke J,Ghadimi BM, et al. Cancer. 2013 Feb 1; 119(3): 522-8.

The PDAC group consisted of 40% early and 60% advanced stage patients.Detection rates were 64% and 85%, respectively. The sensitivity andspecificity of the PAM4 assay was determined for PDAC vs. CP (FIG. 30A)and for PDAC vs. all benign tissue samples (FIG. 30B). The calculatedvalues of AUC were 0.84 and 0.85, respectively.

Approximately 20% of patients with chronic pancreatitis (CP) arepositive by use of the serum-based immunoassay. This issue is criticalto the interpretation of the results with PAM4-positive CP patientsbeing either false positives, or perhaps, the discovery of occultneoplasia. Thus, we undertook an extensive immunohistochemicalevaluation of PAM4-reactivity in CP tissue specimens.

FIG. 31 shows comparative labeling of PDAC vs. non-neoplastic prostatetissue by PAM4 antibody vs. antibodies against MUC1, MUC4, CEACAM6 andCA19-9. Each of the antibodies reacted with PDAC. PAM4 showed noreactivity with normal tissue. The same antibodies were compared in asample showing a PanIN-2 lesion arising within a background of CP, withpartial loss of acinar cells, some fibrosis and PanIN-associatedacinar-ductal metaplasia (ADM) (not shown). No labeling was observedwith PAM4 in any of the tissues within CP, including isolated ADM (notshown). Each of the other antibodies showed some binding tonon-neoplastic tissue (not shown). Table 23 and Table 24 showcomparative results of labeling with PAM4 vs. antibodies against MUC1,MUC4, CEACAM6 and CA19-9.

TABLE 23 Expression of Biomarkers in Pancreatic Ductal AdenocarcinomaPAM4 MUC1 MUC4 CEACAM6 CA19-9 Number 43 43 43 42 43 Focal Labelinga  8(24%)b 1 (2%)  4 (15%) 3 (8%) 2 (5%) Diffuse Labeling 26 (76%) 42 (98%)22 (85%) 35 (92%) 37 (95%) Total Labeled 34 (79%)  43 (100%) 26 (60%) 38(90%) 39 (91%) Adjacent Normal 0 (0%)  14 (100%)  6 (43%)  14 (100%)  14(100%) (N = 14) a-Focal labeling, 5% to 25% of the appropriate tissuecomponents labeled with the indicated MAb; Diffuse, >25% of theappropriate tissue components labeled with the indicated MAb; Total,focal + diffuse. bvalue provided in parenthesis is the percentage oftotal N PDAC specimens evaluated

TABLE 24 Expression of Biomarkers in Chronic Pancreatitis N PAM4 MUC1MUC4 CEACAM6 CA19-9 Chronic 32 Pancreatitis PanIN1  5 2 2 1 5 5 PanIN2 5 4 4 3 5 5 Ducts 32a 0 22 25 31 29 Acinar cells 32a 0 27 8 30 29Isolated 32a 0 24 0 0 26 ADM

We conclude that PAM4 is not reactive with the non-neoplastic tissuesfrom chronic pancreatitis (CP) patients, but rather with PDAC and itsneoplastic precursor lesions, such as PanINs, which are known to developwithin the inflamed parenchyma. Together with results from a priorstudy, we have evaluated a total of 51 specimens of CP, finding that inno instance was PAM4 reactive with the inflamed parenchyma. On the otherhand, each of the other biomarkers investigated, MUC1, MUC4, CEACAM6,and CA19-9, were unable to differentiate PDAC and benign, non-neoplastictissues. These latter biomarkers were expressed to varying extents inCP-associated PanIN lesions, but also in non-neoplastic ducts andisolated ADM. A PAM4-based EIA to quantitate antigen in patient serashows high sensitivity and specificity for detection of PDAC.Approximately ⅔ of patients with stage-1 disease are positive forcirculating PAM4-antigen. We speculate that CP patients (and perhapsothers having disease with high risk for development of PDAC), who arefound to have positive levels of PAM4-reactive antigen in thecirculation, may have occult PDAC and/or significant mass of precursorlesions producing the PAM4-biomarker.

Example 36 Mapping the PAM4 Epitope on MUC5AC

Summary

Indirect and sandwich enzyme immunoassays (EIA) were performed tocompare and contrast the reactivity of PAM4 with several anti-mucinantibodies having known reactivity to specific mucin species (e.g.,MUC1, MUC4, MUC5AC, etc.). Studies designed to block reactivity of PAM4with its specific antigen also were performed. We demonstrated that MAbs2-11M1 and 45M1, each reactive with MUC5AC, are able to provide signalin a heterologous sandwich immunoassay where PAM4 is the captureantibody. Further, we identified MAbs 21M1, 62M1, and 463M1, eachreactive with MUC5AC, as inhibiting the reaction of PAM4 with itsspecific epitope. MAbs directed to MUC1, MUC3, MUC4, MUC16 and CEACAM6were not reactive with PAM4-captured antigen, nor are they able to blockthe reaction of PAM4 with its antigen. We concluded that MUC5AC is themucin species to which PAM4 antibody is reactive.

Background

Mucin glycoproteins are high molecular weight, heavily glycosylated,proteins that include at least 19 species categorized on the basis oftheir unique protein cores. They can be found as either transmembranecomponents of the cell or as secreted products. Abnormal expression ofmucins is a well-known occurrence in many forms of cancer (seeHollingsworth & Swanson, 2004, Nat Rev Cancer 4:45-60; Kufe, 2009, NatRev Cancer 9:874-85; Rachagani et al., 2009, Biofactors 35:509-27),including pancreatic ductal adenocarcinoma (PDAC) (Ringel & Lohr, 2003,Mo lancer 2:9-13; Andrianifahanana et al., 2001, Clin Cancer Res7:4033-40; Torres et al., 2012, Curr Pharm Des 18:2472-81).Neo-expression and/or upregulation/downregulation of specific mucinspecies, with and without the generation of newly transcribed andtranslated splice variants (Schmid, 2003, Oncol Rep 10:1981-85), havebeen well-documented in the literature. Alteration of carbohydratemoieties through the addition of new terminal sugars (e.g., neuraminicacids), underglycosylation, and other abnormal biochemical pathways alsohave been observed (Brockhausen, 2006, EMBO Rep 7:599-604; Yue et al.,2009, Mol Cell Proteomics 8:1697-707; Haab et al., 2010, Ann Surg251:937-45). These modifications may lead to changes in conformationalstructure and/or appearance or disappearance of specific epitopes.Additionally, changes may be observed for the intracellular distributionof the mucin species under consideration, such as MUC1, which in normaltissues is a transmembrane glycoprotein, but with neoplastictransformation is found in the cytoplasm as well (Jass et al., 1995, JPathol 176:143-49; Cao et al., 1997, Virchows Arch 431:159-66). Theseevents may prove to be of biological and clinical significance in theprocess of neoplastic development and progression, as well as providenew biomarkers/targets for early detection and targeted therapy ofcancer.

Our laboratory initially reported the use of a polyclonal antiserum toidentify a pancreatic ductal mucin, which at the level of sensitivityprovided by indirect immunohistochemistry (IHC), was shown to contain anepitope relatively specific to the pancreas (Gold et al., 1983, CancerRes 43:235-38), and ultimately resulted in the development of monoclonalantibody (MAb), PAM4 (Gold et al., 1994, Int J Cancer 57:204-10), alsoknown as clivatuzumab in its humanized form. PAM4 demonstrates highspecificity for PDAC with little to no reactivity towards normal andbenign, non-neoplastic, pancreatic tissues, although it does showlimited reactivity (approximately 10% of all specimens examined) withadenocarcinomas originating in certain other organs (e.g., stomach,colon, lung) (Gold et al., 1994, Int J Cancer 57:204-10; Gold et al.,2007, Clin Cancer Res 13:7380-87; Gold et al., 2010, Cancer EpidemiolBiomarkers Prev 19:2786-94). PAM4 identifies a biomarker that, ifpresent, provides a high diagnostic likelihood of the presence ofpancreatic neoplasia (Gold et al., 2010, Cancer Epidemiol BiomarkersPrev 19:2786-94; Gold et al., 2006, J Clin Oncol 24:252-58; Gold et al.,2013, Cancer 119:522-28). Thus, clinical applications for detection ofearly-stage disease (Gold et al., 2010, Cancer Epidemiol Biomarkers Prev19:2786-94; Gold et al., 2013, Cancer 119:522-28), and antibody-targetedimaging and therapy, are being pursued (Gulec et al., 2011, Clin CancerRes 17:4091-4100; Ocean et al., 2012, Cancer 118:5497-5506). In additionto PDAC, the PAM4-biomarker is expressed in the precursor lesions,pancreatic intraepithelial neoplasia (PanIN, including the earliestdeveloping lesion, PanIN-1A), and intraductal papillary mucinousneoplasia (IPMN), suggesting that there may be oncogenic significance toits expression (Gold et al., 2007, Clin Cancer Res 13:7380-87). In thecurrent study, we investigated the identity of the mucin species towhich this clinically-relevant antibody is reactive, in order tounderstand what role this mucin may play in the development andprogression of pancreatic cancers.

Methods

Antigen and Antibodies—

A mucin containing fraction, designated CPM1, was isolated, as describedpreviously (Gold et al., 2006, J Clin Oncol 24:252-58), from the Capan-1human PDAC xenograft in athymic nude mice. Briefly, this consisted ofhomogenization of the dissected tumor in 0.1M ammonium bicarbonatecontaining 0.5M sodium chloride. Following high-speed centrifugation(20,000 g×45 min), the soluble material was chromatographed on aSEPHAROSE® 4B-CL column, and then eluted with the identical ammoniumbicarbonate-sodium chloride solution. The void volume material wascollected, dialyzed against 0.01M sodium phosphate, pH 7.2, and thenpassed through hydroxyapatite to remove nucleic acids and proteins. Thenon-binding, mucin-containing fraction was again dialyzed extensively toremove salts and used as a source of antigen.

Antibodies used in the current study are listed in Table 25 with cloneand source information. For sandwich and blocking studies, PAM4 wasavailable in both murine (mPAM4) and humanized (hPAM4; clivatuzumab)versions provided by Immunomedics, Inc. (Morris Plains, N.J.). All otherMAbs were murine IgG. Mouse ascites fluids containing MAbs 21M1, 45M1,62M1 and 463M1 were kindly provided by Dr. J. Bara, INSERM, Paris,France. PAM4 antibodies and ascites fluid containing ananti-alpha-fetoprotein antibody, employed as a negative control for theblocking studies (reactive with Hep-G2, hepatoceullar carcinoma cells)were provided by Immunomedics, Inc. (Morris Plains, N.J.). A rabbitpolyclonal anti-CPM1 (Gold et al., 1994, Int J Cancer 57:204-210; Goldet al., 2010, Cancer Epidemiol Biomarkers Prev 19:2786-94) IgG served asthe positive control with detection by a horseradish peroxidase(HRP)-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, WestGrove, Pa.).

TABLE 25 Monoclonal antibodies used Antigen Clone name Source MUC1 MA5Immunomedics MUC1 KC4 Immunomedics MUC1 CM1 Gene Tex MUC2 994/152 AbcamMUC3 M3.1 Abcam MUC3 M3A LifeSpan Bio MUC4 8G7 Santa Cruz Biotech MUC5AC2-11M1 Santa Cruz Biotech MUC5AC 45M1 Santa Cruz Biotech MUC5AC CLH2Santa Cruz Biotech MUC16 X306 Novus Bio MUC16 X325 Abcam CEACAM5 MN14Immunomedics CEACAM6 MN15 Immunomedics CA 19-9 CA 19-9 Santa CruzBiotech Immunomedics, Inc. - Morris Plains, NJ; GeneTex - Irvine, CA;Abcam - Cambridge, MA; LifeSpan Biosciences, Inc. - Seattle, WA; SantaCruz Biotechnology, Inc. - Santa Cruz, CA; Novus Biologicals -Littleton, CO.

Enzyme Immunoassay—

Procedures have been described for both indirect and sandwich enzymeimmunoassays (Gold et al., 1994, Int J Cancer 57:204-210; Gold et al.,2010, Cancer Epidemiol Biomarkers Prev 19:2786-94). For indirectimmunoassays, primary MAbs were used at a concentration of 10 μg/mL toprovide high sensitivity for signal detection. For sandwichimmunoassays, the capture MAb was coated onto the wells at aconcentration of 10 μg/mL, followed by the addition of the CPM1 antigenat various concentrations up to 10 μg/mL. The MAb probe was then addedat a high concentration of 10 μg/mL for detection of response tocaptured antigen. Secondary HRP-labeled anti-species-specific IgG(Jackson ImmunoResearch, West Grove, Pa.) was evaluated initially todetermine optimum concentrations for use in the assay (usually 1:1000 or1:2000). MAb inhibition studies were performed by adding the inhibitingMAb to wells coated with CPM1 antigen, starting at a high concentrationof 100 g/mL of pure MAb or 1:10 dilution of ascites fluid, and titratingto lower amounts. After incubating with the inhibiting antibody at 37°C. for 1 h, the plates were washed, and hPAM4 added to the wells at aconcentration of 0.25 μg/mL. hPAM4 binding was then detected with asecondary probe, HRP-labeled anti-human IgG conjugate.

SDS-PAGE and Western-Blotting—

SDS-PAGE was performed under non-reducing conditions using 4-20%Tris-Glycine gels at 125V for about 2 h. Resolved proteins weretransferred onto a nitrocellulose membrane using the Mini TRANS-BLOT®cell system (Bio-Rad Laboratories, Hercules, Calif.) at 100 V for 1 h.To examine the identity of recombinant proteins, triplicate samples wererun in the same gel and membrane with transferred samples were cut intothree pieces for probing with HRP-anti-Myc, HRP-hPAM4, and 45M1 plusHRP-GAM, respectively. The signals were developed with SUPERSIGNAL™ WestDura Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham,Mass.).

Results

Several MAbs were evaluated by indirect EIA for reactivity with platescoated with CPM1 (FIG. 32), a high molecular weight mucin fractionisolated from the Capan-1 human pancreatic cancer xenograft. Murine PAM4and MAbs reactive specifically with MUC1 and MUC5AC mucins providedelevated reactivity in this indirect immunoassay, with minor reactivityalso observed for MAbs directed to MUC3 and CEACAM6. Essentially noreaction was seen with MAbs to MUC2, MUC4, MUC16, and CEACAM5glycoproteins, or the CA19-9 carbohydrate epitope.

It should be noted that a negative EIA reaction does not necessarilyindicate absence of the mucin-antigen, because the specific epitopestructure may be present, but inaccessible (i.e., cryptic). This islikely the case for MAb-CLH2 anti-MUC5AC generated against a peptidederived from the mucin's tandem repeat (Reis et al., 1997, Int J Cancer74:112-21), since the other two anti-MUC5AC MAbs were highly reactive.Similarly, CM1 anti-MUC1 was considerably less reactive than MA5 and KC4anti-MUC1 antibodies. Capan-1 cells produce well-differentiated tumorswith highly glycosylated mucins. Thus, it is likely that both CLH2 andCM1, reactive with the tandem repeat domains of their respective mucins,would not be reactive with CPM1, since the tandem repeat epitopes areinaccessible.

We then evaluated whether the anti-mucin MAbs were reactive withPAM4-captured mucin. Humanized PAM4 (hPAM4)-coated plates were used tocapture the specific mucin-antigen from the CPM1 fraction, which wasthen probed with various anti-mucin MAbs. Murine MAbs (mMAbs)specifically reactive with MUC1, MUC3, MUC4, MUC16 and CEACAM6 did notprovide a signal in these heterologous sandwich immunoassays (notshown). On the other hand, both anti-MUC5AC mMAbs tested, 45M1 and2-11M1, gave positive reactions with the hPAM4-captured antigen (FIG.33), with 45M1 showing significantly greater reaction than 2-11M1(Kd=14.32±1.08 μg/mL and 24.4±7.83 μg/mL, respectively, for MAbs 45M1and 2-11M1; P<0.001). However, neither of these individual anti-MUC5ACMAbs provided as strong signal intensity as the rabbit anti-CPM1polyclonal IgG fraction. Importantly, mPAM4 did not bind to thehPAM4-captured antigen, nor did hPAM4 bind to mPAM4-captured antigen,suggesting that the PAM4 epitope is present at low density, possiblyonly a single site within the mucin-antigen.

Follow-up studies were designed to inhibit the binding of hPAM4 toCPM1-coated plates (FIG. 34A-B). Although 2-11M1 anti-MUC5AC was unableto inhibit hPAM4-CPM1 binding, 45M1 anti-MUC5AC was able to provide alimited inhibitory effect, with IC_(max)=25.5% inhibition (FIG. 34A).mPAM4, included as a positive control, provided IC_(max)=92.4%self-inhibition at a concentration 0.1 μg/mL, while the MA5 and KC4anti-MUC1 antibodies provided no inhibition, even at the highestconcentration evaluated (10 μg/mL) (FIG. 34A). hPAM4 was unable tocompletely block mPAM4 binding to the CPM1 antigen (IC_(max)=52.8%) (notshown), a not unexpected finding since the humanized version of PAM4 isknown to have a lower affinity than the murine parent. Ascites fluidscontaining mMAbs with known mapping to MUC5AC were serially diluted asinhibitory reagents, with results shown in FIG. 34B. mMAbs 21M1, 62M1,and 463M1 each provided inhibition similar to the results shown formPAM4 self-blocking, with 45M1 ascites providing limited inhibition,similar to what was observed with the commercially available 45M1-IgG(FIG. 34B). Ascites fluid containing a murine anti-alpha-fetoprotein(AFP), included here as a negative control, provided no inhibition ofthe hPAM4 binding to CPM1 (FIG. 34B). Unfortunately, insufficientvolumes of ascites precluded determination of MAb concentrations, sothat relative blocking efficiency could not be calculated.

Discussion

The current Example suggests that PAM4 is reactive with the MUC5AC mucinglycoprotein. FIG. 35 presents a map of the MUC5AC mucin domains withreactive epitopes indicated for several of the anti-MUC5AC MAbs employedin our studies (Nollet et al., 2002, Int J Cancer 99:336-43; Nollet etal., 2004, Hybrid Hybridomics 23:93-99; Lidell et al., 2008, FEBS J275:481-89). CLH2 is reactive with the peptide core of the tandem repeatdomain (Reis et al., 1997, Int J Cancer 74:112-21), and is likely acryptic epitope within the Capan-1 tumor-derived MUC5AC. 2-11M1 isreactive with the N-terminus of the mucin (Nollet et al., 2004, HybridHyridomics 23:93-99), and 45M1 at the furthest N-terminal region of thecysteine-rich, C-terminus (Lidell et al., 2008, FEBS J 275:481-89). Bothof these MAbs were reactive with PAM4-captured mucin, whereas MAbs toMUCs 1, 3, 4, and 16 were not. We observed that 45M1 provides asignificantly greater signal response than 2-11M1, suggesting a greaterdensity of 45M1-epitopes than 2-11M1-epitopes within CPM1. However, thismay simply be due to a loss of 2-11M1 epitopes through proteolyticdigestion of the relatively non-glycosylated N-terminus, and/ormolecular shear of this very large glycoprotein during purification. Inany case, the 2-11M1 antibody provided no inhibition of the hPAM4-CPM1interaction, suggesting the epitope is located distant to thePAM4-epitope.

On the other hand, 45M1 did inhibit the hPAM4-CPM1 interaction, albeitonly partially, suggesting that the PAM4-epitope is within theC-terminal region of the mucin or conformationally altered byinteraction of this antibody with the mucin molecule. MAbs 21M1, 62M1,and 463M1 have also been mapped to the C-terminal region of the MUC5ACmucin (Nollet et al., 2002 Int J Cancer 99:336-43; et al., 2004, HybridHybridomics 23:93-99; Lidell et al., 2008, FEBS J 275:481-89), and eachprovided significant inhibition of the PAM4-mucin reaction. Takentogether, our data provide direct evidence that PAM4 is reactive withthe identical mucin (MUC5AC), and that the PAM4 epitope is eitherdirectly-blocked, or conformationally modified, by interaction of theseMAbs with the MUC5AC antigen.

We had initially reported that PAM4 was reactive with the MUC1 mucinspecies (Gold et al., 2007, Clin Cancer Res 13:7380-87; Gold et al.,2006, J Clin Oncol 24:252-58). This was based upon MUC1-genetransfection studies, whereby PAM4 was observed to react with thegene-transfected, MUC1⁺ cell line, but not the MUC1⁻ parental cell lineor vector control cell lines. However, other evidence acquired sincethen has questioned this interpretation, suggesting that MUC1transfection may have upregulated other mucins as well. Prior resultsfrom our laboratory lend support to the current findings. The PAM4epitope was found to be highly sensitive to mild reduction withdithiothreitol (0.02M, 15 min, 20° C.) or heat (100° C., 2 min),suggesting the epitope is peptide in nature, and highly dependent upon aspecific conformation of the protein core kept intact by disulfidebridges (Gold et al., 1994, Int J Cancer 57:204-10). This is unlikely tobe MUC1 with all of the cysteines located within the transmembranedomain of the mucin, but is consistent with the loss of reactivity shownby several anti-MUC5AC MAbs upon reduction of the mucin antigen.Further, employing immunohistochemical methods, we reported thatfrequency of expression and morphologic distribution of the PAM4-epitopewithin PDAC and its precursor lesions shared greater similarity to thosedescribed for MUC5AC than for MUC1 (Gold et al., 2007, Clin Cancer Res13:7380-87).

In conclusion, antibodies that bind to the PAM4 epitope of MUC5AC are ofuse for detection and differential diagnosis of pancreatic cancer.Immunoconjugates of such antibodies are of use for pancreatic cancertherapy.

Example 37 DOTA Conjugates of PAM4

The hPAM4 antibody was prepared as described in Example The genes ofCDR-grafted V_(H) and Vκ chains of hPAM4 were inserted into the pdHL2plasmid vector, a DHFR-based amplifiable expression system. The plasmidwas transfected into the murine myeloma cell line, Sp2/0-Ag14 (ATCC,Manassas, Va.) to generate the cell clones producing hPAM4. The completemature amino acid sequence is shown below.

hPAM4 Heavy Chain (SEQ ID NO: 120)QVQLQQSGAEVKKPGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYINPYNDGTQYNEKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hPAM4 light chain (SEQID NO: 121) DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

The DNA and amino acid sequences of hPAM4 Vκ and V_(H) are shown in FIG.4A and FIG. 4B, respectively, with the CDRs identified in bold andunderlined.

The current cell clone name is hPAM4-2E3 and is produced in Sp2/0 hostcells, DHFR expression system. The antibody is a humanized IgG_(1κ)glycoprotein. A glycosylation site on the heavy chain (Asn299) has acomposition per mole of hPAM4-DOTA: 0.5 Fuc, 6.3 GlcNAc, 6.3 Man, 0.3Gal and 0.15 Neu5Gc; glycosylation species: G0F 70%, G1F 23%, G2F 2%,G1FS1 4%, G2FS1 1%. There are 16 S—S bonds (32 SH), identified andlocated exactly as theoretical prediction based on the above sequence.

An hPAM4-DOTA product was prepared from purified hPAM4 IgG that wascoupled with the 12-membered macrocyclic chelating agent1,4,7,10-tetraazacyclododecane-N, N′,N″,N′″-tetraacetic acid (DOTA).

DOTA was conjugated via one of the carboxyl moieties to reactive siteson the hPAM4 antibody to generate a stable conjugate. The coupling isassumed to be via stable amide bond to the antibody's lysine side-chainamino group.

The chemical conjugation was performed by first reacting DOTA withN-hydroxysulfo-succinimide (sulfo-NHS) in the presence of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to generateactivated DOTA, then incubating activated DOTA with purified hPAM4antibody. Conditions were optimized to yield a substitution ratio of 4-7DOTA moieties per antibody molecule, as determined by mass spectrometryassays.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

What is claimed is:
 1. A method of treating a cancer that expressesMUC5ac comprising administering to a human subject with a cancer thatexpresses MUC5ac a humanized anti-MUC5ac monoclonal antibody orantigen-binding fragment thereof that binds to an epitope located withinthe second cysteine-rich domain (Cys2, amino acid residues 1575-1725) ofMUC5ac, wherein the anti-MUC5ac antibody is conjugated to at least onetherapeutic agent.
 2. The method of claim 1, wherein the cancer isselected from the group consisting of gastric, colorectal, lung, biliaryand pancreatic adenocarcinoma.
 3. The method of claim 1, wherein thecancer is pancreatic adenocarcinoma.
 4. The method of claim 1, whereinthe therapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an anti-sense oligonucleotide, siRNA, an enzyme inhibitor, aphotoactive therapeutic agent, a cytotoxic agent, a drug, a toxin, anangiogenesis inhibitor and a pro-apoptotic agent.
 5. The method of claim4, wherein the radionuclide is selected from the group consisting of¹⁴C, ¹³N, ¹⁵O, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru,⁹⁷Ru, ⁹⁹Mo, ^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd,¹⁰⁹Pt, ¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te, ^(122m)Te, ¹²⁵I,^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho,¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁹⁹Au,²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po, ²¹⁷At,²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²⁵⁵Fm and Th²²⁷.
 6. The method ofclaim 5, wherein the radionuclide is ⁹⁰Y.
 7. The method of claim 4,wherein the drug is selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX, cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 8. The method ofclaim 4, wherein the toxin is elected from the group consisting ofricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, ranpirnase,gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonasendotoxin.
 9. The method of claim 4, wherein the immunomodulator isselected from the group consisting of a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), erythropoietin, thrombopoietin tumornecrosis factor (TNF), granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, human growth hormone,N-methionyl human growth hormone, bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, thrombopoietin (TPO),NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growthfactor-I, insulin-like growth factor-II, erythropoietin (EPO),macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin,endostatin, and lymphotoxin.
 10. The method of claim 4, wherein thetherapeutic agent is a tyrosine kinase inhibitor selected from the groupconsisting of canertinib, dasatinib, erlotinib, gefitinib, imatinib,lapatinib, leflunomide, nilotinib, pazopanib, semaxinib, sorafenib,sunitinib, sutent, vatalanib, PCI-32765 (ibrutinib), PCI-45292,GDC-0834, LFM-A13 and RN486.
 11. The method of claim 1, furthercomprising administering at least one other therapeutic agent to thehuman subject, wherein the at least one other therapeutic agent isselected from the group consisting of a second antibody, a secondantigen-binding antibody fragment, an immunoconjugate, animmunomodulator, a hormone, a hormone antagonist, an enzyme, ananti-sense oligonucleotide, siRNA, an enzyme inhibitor, a photoactivetherapeutic agent, a cytotoxic agent, a drug, an angiogenesis inhibitorand a pro-apoptotic agent.
 12. The method of claim 11, wherein thesecond antibody, second antigen-binding antibody fragment, orimmunoconjugate binds to an antigen selected from the group consistingof CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, Le^(a), Le(y), CEACAM5,CEACAM6, CSAp, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, HLA-DR,CD40, CD74, CD138, HER2/neu, EGFR, EGP-1, EGP-2, VEGF, PlGF,insulin-like growth factor, tenascin, platelet-derived growth factor,IL-6, bcl-2, K-ras, p53 and cMET.
 13. The method of claim 11, whereinthe second antibody, second antigen-binding antibody fragment, orimmunoconjugate is selected from the group consisting of hR1(anti-IGF-1R), hPAM4 (anti-MUC5ac), hIMMU-31 (anti-AFP), hLL1(anti-CD74), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hL243 IgG4P(anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7(anti-EGP-1 or anti-TROP-2), hMN-3 (anti-CEACAM6), Ab124 (anti-CXCR4)and Ab125 (anti-CXCR4).
 14. A method of treating a cancer that expressesMUC5ac comprising: a. administering to a human subject with a cancerthat expresses MUC5ac a bispecific antibody comprising (i) a humanizedanti-MUC5ac monoclonal antibody or antigen-binding fragment thereof thatbinds to an epitope located within the second cysteine-rich domain(Cys2, amino acid residues 1575-1725) of MUC5ac, and (ii) an anti-haptenantibody or antigen-binding fragment thereof; and b. administering tothe individual a targetable construct comprising at least one copy ofthe hapten, wherein the targetable construct is conjugated to at leastone therapeutic agent.
 15. The method of claim 14, wherein the hapten isHSG or In-DTPA.
 16. The method of claim 14, wherein the cancer isselected from the group consisting of gastric, colorectal, lung, biliaryand pancreatic adenocarcinoma.
 17. The method of claim 14, wherein thecancer is pancreatic adenocarcinoma.
 18. The method of claim 14, whereinthe therapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an anti-sense oligonucleotide, siRNA, an enzyme inhibitor, aphotoactive therapeutic agent, a cytotoxic agent, a drug, a toxin, anangiogenesis inhibitor and a pro-apoptotic agent.
 19. The method ofclaim 18, wherein the radionuclide is selected from the group consistingof ¹⁴C, ¹³N, ¹⁵O, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br, ⁸⁹Sr, ⁹⁰Y,⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo, ^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg,¹⁰⁹Pd, ¹⁰⁹Pt, ¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te, ^(122m)Te,¹²⁵I, ^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm,¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au,¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po,²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²⁵⁵Fm and Th²²⁷.
 20. Themethod of claim 18, wherein the radionuclide is ⁹⁰Y.
 21. The method ofclaim 18, wherein the drug is selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX, cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.
 22. The method ofclaim 18, wherein the toxin is elected from the group consisting ofricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. 23.The method of claim 18, wherein the immunomodulator is selected from thegroup consisting of a cytokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), erythropoietin, thrombopoietin tumor necrosisfactor (TNF), granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, human growth hormone,N-methionyl human growth hormone, bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, thrombopoietin (TPO),NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growthfactor-I, insulin-like growth factor-II, erythropoietin (EPO),macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin,endostatin, and lymphotoxin.
 24. The method of claim 18, wherein thetherapeutic agent is a tyrosine kinase inhibitor selected from the groupconsisting of canertinib, dasatinib, erlotinib, gefitinib, imatinib,lapatinib, leflunomide, nilotinib, pazopanib, semaxinib, sorafenib,sunitinib, sutent, vatalanib, PCI-32765 (ibrutinib), PCI-45292,GDC-0834, LFM-A13 and RN486.